Hbv epitope reactive exogenous t cell receptor (tcr) and uses thereof

ABSTRACT

There is provided at least one isolated cell comprising at least one HBV epitope-reactive exogenous T cell receptor and/or fragment thereof, and methods for producing them. In particular, there is provided polynucleotides, constructs and vectors encoding at least one HBV epitope-reactive exogenous T cell receptor for use in the treatment of Hepatitis B Virus (HBV) and Hepatocellular Carcinoma (HCC). The invention further provides kits and methods of detection of HBV and HCC.

FIELD OF THE INVENTION

The present invention relates generally to the field of immune mediatedtherapies for treatment of Hepatitis B Virus (HBV) and HBV-relateddiseases such as cirrhosis and Hepatocellular carcinoma (HCC) and tomethods of preparing these therapies. In particular, the immune mediatedtherapies may be in the form of cells expressing at least one HBVepitope-reactive exogenous T cell receptor and/or polynucleotides,vectors and/or polypeptides encoding the HBV epitope-reactive T cellreceptor.

BACKGROUND OF THE ART

Hepatitis B virus (HBV) infects the liver of hominoidae, includinghumans, and causes an inflammation called hepatitis. It is a DNA virusand one of many unrelated viruses that cause viral hepatitis. HBVcurrently infects at least 350 million people worldwide. 75% ofchronically infected HBV patients are living in Asia but the virus has aworld-wide diffusion. For example, in the United States, approximately1.5 million Americans, or about 0.5% of the population have HBV. Theinfection is especially more common in certain risk groups like men whohave sex with other men, renal dialysis patients and persons withhaemophilia. Chronic HBV infections affects 10 to 15% of firstgeneration Asian Americans and approximately 5% of children adopted fromRussia, Asia and Eastern Europe have chronic HBV infections. Chronic HBVinfections may eventually lead to liver cirrhosis and Hepatocellularcarcinoma (HCC), a fatal disease with very poor response to currentchemotherapy.

Currently available methods of treatment for this global large reservoirof chronically infected subjects are challenging as existing drugssuppress but do not eliminate HBV. Though majority of HBV infectedpatients respond to currently available methods of treatment showingimprovements in liver histology and serum Alanine transaminase (ALT)levels, almost all patients relapse when treatment is stopped.Furthermore, the more common methods of treatment of HBV involve the useof drugs such as lamivudine and adefovir. These drugs however, result indevelopment of anti-viral resistance in the patients, which occur inapproximately 20% of patients treated with lamivudine and in about 3% ofthose treated with adefovir each year. Ultimately a large proportion ofpatients would develop resistance, at which point, the anti-viral drugswould have little effect.

There is thus a global need for an effective method of anti-viraltherapy that results in an HBV specific immune response to efficientlyand successfully eliminate the covalently closed circular form of HBV ina patient. Naturally occurring HBV-epitope specific T cells found insome subjects, lead to the subjects having an efficient and innatecontrol of the HBV infection. Past studies have shown that patients withpersistent/chronic HBV infection have their HBV-epitope specific T cellsdeleted or functionally altered. Therefore, increased knowledge ofvirus-host interactions during HBV infection has prompted speculationthat therapeutic restoration of the defective anti-viral immunitypresent in patients with chronic infection could possibly lead todisease resolution. The validity of this concept was directlydemonstrated in patients with chronic HBV infection who underwent bonemarrow transplantation and received marrows from donors with naturalimmunity to HBV. Infusion of a healthy HBV-primed immune system led toresolution of chronic HBV infection in these patients. However, bonemarrow transplantation is clearly not an easy therapeutic option forchronically HBV infected patients and attempts to boost HBV-specificimmunity using various vaccines in patients with chronic hepatitis Bhave been disappointing.

A potential cause of failure of the therapeutic vaccine strategy is thefact that the immune system of HBV chronic carriers does not have thesame efficiency and repertoire of specificities as that of healthynon-HBV infected subjects.

Further, persistent high production of viral antigens in chronic HBVinfected patients can delete or tolerize antigen-specific T cells.Chronic HBV infected patients are thus characterized by low/absentHBV-specific CD4+ and CD8+ T cell responses. Moreover, it has beenspeculated that both low T cell avidity and an ineffective cytokineprofile generated in response to HBV infection may contribute to thedevelopment of chronic HBV infection rather than viral clearance.

Clearly, more effective therapies targeting molecules involved in thecytokine profile of HBV infected patients are necessary in order toreduce the worldwide morbidity and mortality from HBV infection andHBV-related malignancies.

SUMMARY OF THE INVENTION

The present invention addresses the problems above, and in particularprovides a novel, efficient and effective method of treating HBVinfection by redirecting the specificity of lymphocytes of chronic HBVinfected patients using exogenous T cell receptor (TCR) transfer.

According to a first aspect, the present invention provides at least oneisolated cell comprising at least one HBV epitope-reactive exogenous Tcell receptor (TCR) and/or fragment thereof.

In particular, the HBV epitope may be HLA-A2 restricted. More inparticular, the HBV epitope may comprise at least one hepatitis B coreantigen or one hepatitis B envelope antigen, or a mutant thereof. Evenmore in particular, the HBV epitope may comprise HBc18-27, HBs370-79and/or a mutant thereof. The HBc18-27 epitope may comprise at least onesequence selected from the group consisting of SEQ ID NOs:25 to 39. Inparticular, the HBc18-27 epitope may comprise sequence, SEQ ID NO:25.The HBs370-79 epitope may comprise at least one sequence selected fromthe group consisting of SEQ ID NO:56 to 58. In particular, the HBs370-79epitope may comprise sequence SEQ ID NO:56.

In particular, the cell according to the present invention may furthercomprise at least one second HBV epitope-reactive exogenous TCR and/orfragment thereof, wherein the second HBV epitope may be different fromthe first HBV epitope. In particular, the first HBV epitope may beHBc18-27 and the second epitope may be HBs370-79.

The exogenous TCR may comprise at least one α-chain comprising at leastone amino acid sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10 and SEQ ID NO:11 or mutants thereof. In particular,the exogenous TCR comprises at least one α-chain comprising thesequences SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. The exogenous TCRmay comprise at least one β-chain comprising at least one amino acidsequence selected from the group consisting of SEQ ID NO:21, SEQ IDNO:22 and SEQ ID NO:23 or mutants thereof. More in particular, theexogenous TCR may comprise at least one β-chain comprising the sequencesSEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. All sequences are shown inTables 1 and 2.

In particular, the exogenous TCR may comprise at least one α-chainhaving at least 80% amino acid identity to SEQ ID NO:12 or a fragmentthereof and/or at least one β-chain having at least 80% amino acididentity to SEQ ID NO:24 or a fragment thereof. More in particular, theexogenous TCR may comprise at least one α-chain of SEQ ID NO:12 and/orat least one β-chain of SEQ ID NO:24.

The exogenous TCR may comprise at least one α-chain comprising at leastone amino acid sequence selected from the group consisting SEQ ID NO:44,SEQ ID NO:45 and SEQ ID NO:46 or mutants thereof. In particular, theexogenous TCR comprises at least one α-chain comprising the sequencesSEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46. The exogenous TCR maycomprise at least one β-chain comprising at least one amino acidsequence selected from the group consisting of SEQ ID NO:52, SEQ IDNO:53 and SEQ ID NO:54 or mutants thereof. More in particular, theexogenous TCR may comprise at least one β-chain comprising the sequencesSEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54. All sequences are shown inTables 1 and 2.

In particular, the exogenous TCR may comprise at least one α-chainhaving at least 80% amino acid identity to SEQ ID NO:47 or a fragmentthereof and/or at least one β-chain having at least 80% amino acididentity to SEQ ID NO:55 or a fragment thereof. More in particular, theexogenous TCR may comprise at least one α-chain of SEQ ID NO:47 and/orat least one β-chain of SEQ ID NO:55.

In particular, the cell according to the present invention may be atleast one hematopoietic stem cell. More in particular, the cell may beat least one Peripheral Blood Lymphocytes (PBL)-derived T cell.

According to another aspect, the present invention provides at least oneisolated polynucleotide comprising at least one sequence encoding atleast one α-chain and/or at least one sequence encoding at least oneβ-chain wherein, the α-chain and β-chain may be part of at least oneexogenous HBV epitope-reactive TCR. The HBV epitope may be HBc18-27and/or HBs370-79.

In particular, the sequence encoding the α-chain comprises at least onesequence selected from SEQ ID NO:1 and SEQ ID NO:5, at least onesequence selected from SEQ ID NO:2 and SEQ ID NO:6 and at least onesequence selected from SEQ ID NO:3 and SEQ ID NO:7 and/or the sequenceencoding the β-chain comprises at least one sequence selected from SEQID NO:13 and SEQ ID NO:17, at least one sequence selected from SEQ IDNO:14 and SEQ ID NO:18 and at least one sequence selected from SEQ IDNO:15 and SEQ ID NO:19. More in particular, the sequence encoding theα-chain may comprise SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 and/or thesequence encoding the β-chain may comprise SEQ ID NO:17, SEQ ID NO:18and SEQ ID NO:19.

In particular, the α-chain of the HBV epitope-reactive exogenous TCR mayhave at least 80% sequence identity to SEQ ID NO:4 or SEQ ID NO:8 and/orthe β-chain of the HBV epitope-reactive exogenous TCR may have at least80% sequence identity to SEQ ID NO:16 or SEQ ID NO:20. More inparticular, the sequence encoding the α-chain may be selected from thegroup consisting of SEQ ID NO:4 and SEQ ID NO:8, and/or the sequenceencoding the β-chain may be selected from the group consisting of SEQ IDNO:16 and SEQ ID NO:20. More in particular, the sequence of the α-chainof the HBc18-27 epitope reactive exogenous TCR may be selected from thegroup consisting of SEQ ID NO:4 and SEQ ID NO:8, and/or the sequence ofthe β-chain of the HBc18-27 epitope reactive exogenous TCR may beselected from the group consisting of SEQ ID NO:16 and SEQ ID NO:20.

In particular, the sequence encoding the α-chain comprises SEQ ID NO:40,SEQ ID NO:41 and SEQ ID NO:42 and/or the sequence encoding the β-chaincomprises SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. More inparticular, the α-chain of the HBV epitope-reactive exogenous TCR mayhave at least 80% sequence identity to SEQ ID NO:43 and the β-chain ofthe HBV epitope-reactive exogenous T cell receptor has at least 80%sequence identity to SEQ ID NO:51. Even more in particular, the sequenceencoding the α-chain comprises SEQ ID NO:43, and/or the sequenceencoding the β-chain comprises SEQ ID NO:51. More in particular, thesequence of the α-chain of the HBs370-79 epitope reactive exogenous TCRmay comprise SEQ ID NO:43 and/or the sequence of the β-chain of theHBs370-79 epitope reactive exogenous TCR may comprise SEQ ID NO:51.

According to another aspect, the present invention provides at least oneisolated polypeptide encoded by at least one polynucleotide of thepresent invention.

According to still another aspect, the present invention provides atleast one isolated polypeptide comprising at least one sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11and at least one further sequence selected from the group consisting ofSEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In particular, the sequencemay have at least 80% amino acid identity to SEQ ID NO:12 and thefurther sequence may have at least 80% amino acid identity to SEQ IDNO:24.

The present invention also provides at least one isolated polypeptidecomprising at least one sequence selected from the group consisting ofSEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46 and/or at least one furthersequence selected from the group consisting of SEQ ID NO:52, SEQ IDNO:53 and SEQ ID NO:54. In particular, the sequence may have at least80% amino acid identity to SEQ ID NO:47 and the further sequence mayhave at least 80% amino acid identity to SEQ ID NO:55.

The polypeptide according to the present invention may be at least oneHBV epitope-reactive exogenous TCR. In particular, the HBV epitope maybe HBc18-27, HBs370-79 and/or a mutant thereof. More in particular, thepolypeptide may be at least one soluble TCR or a fragment thereof. Evenmore in particular, the soluble TCR or fragment thereof may be linked toat least one anti-viral drug.

According to one aspect, the present invention provides at least oneconstruct comprising the polynucleotide of the present inventionoperably connected to at least one promoter.

According to another aspect, the present invention provides at least onevector comprising the construct according to the present invention orthe polynucleotide according to the present invention.

According to still another aspect, the present invention provides atleast one T cell comprising the vector or the construct according to thepresent invention.

According to one aspect, the present invention provides at least onemethod of preparing at least one T cell comprising at least one HBVepitope-reactive exogenous TCR for delivery to at least one subject themethod comprising transducing at least one T cell isolated from thesubject with the construct of or the vector of the present invention.

According to another aspect, the present invention provides at least onemethod of preparing at least one HBV epitope-reactive exogenous TCRcomprising:

-   -   (a) isolating at least one HBV-epitope reactive T cell from at        least one HBV-exposed individual that resolved HBV infection;    -   (b) cloning at least one polynucleotide sequence encoding at        least one α- and/or β-chain of at least one TCR cell receptor        from the HBV-epitope reactive T cell of step (a);    -   (c) delivering the polynucleotide sequence of step (b) to at        least one cell; and    -   (d) incubating the cell under conditions suitable for expression        of the HBV epitope-reactive exogenous T cell receptor by the        cell.

In particular, the method of preparing at least one HBV epitope-reactiveexogenous TCR comprises the isolation of at least one T cell, step (a)which may be HBc18-27 epitope reactive and/or HBs370-79 epitopereactive.

According to one aspect, the present invention provides at least onecell according to the present invention, for use in the treatment of HBVinfection and/or HBV-related hepatocellular carcinoma.

According to another aspect, the present invention provides at least onemethod of treating HBV and/or inhibiting reactivation of HBV in at leastone subject comprising administering to the subject at least oneimmunotherapeutically effective amount of cells, at least one vectorand/or at least one polypeptide according to any aspect of the presentinvention.

According to still another aspect, the present invention provides atleast one method of treating HBV-related hepatocellular carcinoma in atleast one subject comprising administering to the subject at least oneimmunotherapeutically effective amount of cells, at least one vectorand/or at least one polypeptide according to any aspect of the presentinvention.

According to one aspect, the present invention provides at least one invitro method for diagnosing at least one subject that is able to resolveHBV infection, the method comprising:

-   -   (a) providing at least one sample from at least one subject;    -   (b) detecting the presence of at least one polynucleotide        comprising, substantially consisting of or consisting of at        least one nucleic acid sequence selected from SEQ ID NO: 4, SEQ        ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 43, SEQ ID        NO: 51, a homologue and/or a fragment thereof; and/or    -   (c) detecting the presence of at least one polypeptide        comprising, substantially consisting of or consisting of the        amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO:        47, SEQ ID NO: 55, a homologue and/or a fragment thereof;        wherein the presence of the polynucleotide and/or the        polypeptide is indicative of the subject being able to resolve        the HBV infection.

According to yet another aspect, the present invention provides at leastone in vitro method for diagnosing at least one subject as having or asbeing at risk of having HBV infections and/or HBV related hepatocellularcarcinoma, the method comprising:

-   -   (a) providing at least one sample from at least one subject;    -   (b) detecting the presence of at least one polynucleotide        comprising, substantially consisting of or consisting of at        least one nucleic acid sequence selected from SEQ ID NO: 4, SEQ        ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 43, SEQ ID        NO: 51, a homologue and/or a fragment thereof; and/or    -   (c) detecting the presence of at least one polypeptide        comprising, substantially consisting of or consisting of the        amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO:        47, SEQ ID NO: 55, a homologue and/or a fragment thereof;        wherein the absence of the polynucleotide and/or the polypeptide        correlates with the likelihood of the subject as having or as        being at risk for having HBV-infection and/or HBV-related        hepatocellular carcinoma.

According to another aspect, the present invention provides at least oneuse of at least one cell, at least one vector and/or at least onepolypeptide according to any aspect of the present invention in thepreparation of a medicament for treating HBV infection and/orHBV-related hepatocellular carcinoma.

According to still another aspect, the present invention provides atleast one kit for detecting and/or treatment of HBV infection and/orHBV-related hepatocellular carcinoma, the kit comprising at least onecell, at least one vector and/or at least one polypeptide according toany aspect of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram of the steps involved in producing acell comprising at least one HBV epitope-reactive exogenous T cellreceptor. It comprises the isolation of a T cell clone from an HLA-A2positive HBV patient who has resolved the infection, using the T cellclone to form a retroviral vector construct encoding HBV T cell receptorand using the vector to form cells that express the HBV epitope-reactiveexogenous T cell receptor. FIG. 1 was partly taken from Schumacher etal., 2002.

FIG. 2 (A) depicts FACS analysis of peripheral blood lymphocytes (PBL)which were stimulated with HBc18-27 peptide to generate T cell lines.The HBc18-27 specific T cell line was isolated using specificHBc18-27-HLA-A201 pentamers and magnetic separation. The isolatedHBc18-27 specific T cell line was stained with HBc18-27-HLA-A201specific pentamers to confirm clonality. The results show that 100% ofthe T cells were CD8+ and HLA-A2 pentamer positive, indicatingclonality.

FIG. 2 (B) shows a graph with the results of V beta typing of apotential T cell clone, C183, which was derived from a resolved HLA-A201HBV patient using staining with a panel of TCR V beta monoclonalantibodies (MAbs). MAbs reacting with the human TCR V beta region arealmost as specific as a private idiotypic MAb in identifying T cellclones. This method was thus used to evaluate expression of V beta genefamilies. The clone, C183, which was found to be highly specific to HBVcore 18-27 (HBc18-27) epitope. The results suggest that all T cells wereVβ8 positive.

FIG. 3 shows the cytotoxic function of HBc18-27 specific Cytotoxic Tlymphocyte (CTL) clone C183. CarboxyFluoroscein Succinimidyl Ester(CFSE) labelled HepG2 cells pulsed with HBc18-27 (8-10 M) epitope weremixed with HepG2 cells that were not HBc18-27 pulsed. The HepG2 cellswere cultured in the presence or absence of CTL clones at a 1:1 ratiofor 5 h. Cytotoxicity was determined by disappearance of CFSE labelledtargets in the presence of C183 clone (+C183). After 5 h co-incubation,the results show that only CFSE negative cells (no HBc18-27 peptide)remained and nearly all HBc18-27 positive, CFSE positive HepG2 cellswere eliminated by C183, indicating the T cell clone is capable ofkilling tumour cells presenting HBV antigens like HBc18-27. There was nosignificant difference in the cell count when no CTL was added (NO CTL).

FIG. 4 shows that C183 T cell clone recognizes endogenously processedviral antigen presented by hepatocellular carcinoma cells.

FIG. 4 (A) shows the results of HBc18-27 specific Cytotoxic T lymphocyte(CTL), C183, incubated with parental, vector only, HepG2 cell line(HepG2TA2-7) for 5 h as a control. The control was tested for cytokinerelease and cytotoxic degranulation (CD107a+ staining), which revealedthat C183 did not respond to the control cell line HepG2TA2-7 as therewas no activation to degranulate (CD107a+) and did not produce IFN-γ.

FIG. 4 (B) shows that C183 T cell clone activation after co-culture withHepG2.105 cells which expressed different levels of the HBV antigenstimulated IFN-γ production and degranulation, indicating the C183 Tcell clone recognizes the HBc18-27 epitope processed and presented byhepatocellular carcinoma cells. 84% of T cell clones were activatedafter 5 h co-culture with HepG2 cells expressing HBV.

FIG. 5 shows the results of how C183 T cell clone responds to naturallyinfected hepatocytes. Primary hepatocytes which were isolated fromHLA-A2+ or HLA-A2− explanted chronic HBV livers were co-cultured withC183. HBc18-27-specific C183 T cell clone degranulation and IFN-γproduction was measured after incubation of C183 alone, with HLA-A2+uninfected, with HLA-mismatch HBV infected or with HLA-A2+HBV infectedprimary human hepatocytes. C183 activation (12% CD107a+) was onlyobserved following incubation with HLA-A2+HBV infected primary humanhepatocytes indicating that the C183 T cell clone recognizesendogenously processed viral antigen presented by HBV infected primaryhepatocytes that express the correct HLA molecules on their cellsurface.

FIG. 6 shows the ability of T cell clone, C183, to recognize theHBc18-27 epitope from HBV genotypes B and C (grey bars, Asia virus;C18-I) and HBV genotypes A and D (black bars, European/American virus;C18-V) presented by different subtypes of the HLA-A2 MHC class I family.Epstein-Barr virus (EBV) transformed B cells with known subtypes ofHLA-A2 were loaded with increasing concentrations of HBc18-27 epitopefrom different genotypes of HBV and co-cultured with C183 T cell clonefor 5 h. IFN-γ production was measured to determine T cell activation.Data shows that (A) HLA-A201; (B) HLA-A206; and (C) HLA-A207 MHC class Imolecules could present the HBV epitope from all genotypes and, wererecognized by C183 and thus stimulated efficient IFN-γ production by theT cells. C183 T cell clone was found to be able to recognize theHBc18-27 epitope with high sensitivity (≦1 pM) from genotypes B and C orgenotypes A and D and the epitope was recognized almost equally whenpresented by the three most dominant HLA-A2 subtypes A0201 (A), A0206(B) and A0207 (C).

FIG. 7 shows the analysis of T Cell Receptor (TCR) transductionefficiency.

FIG. 7 (A) shows the FACS analysis of peripheral blood lymphocytes (PBL)when they were mock transduced (negative control) or transduced with theHBc18-27 A201 TCR comprised of Vβ8.2 and Vα3 chains. PBL were stainedfor Vβ8 chain 3 days after transduction to determine expression of theintroduced HBc18-27 TCR. Mock transduced PBL expressed endogenous levelsof Vβ8 (CD8+=1.8%; CD4+=2.1). Expression of Vβ8 increased substantiallyafter transduction with HBc18-27 A201 TCR compared to mock transducedcells, which expressed only endogenous levels of Vβ8, indicatingsuccessful transduction and expression.

FIG. 7 (B) shows the results obtained when mock or HBc18-27 TCRtransduced cells were stained with HBc18-27-A201 specific pentamer todetermine if the Vα3 chain was expressed and properly paired with Vβ8.2chain on the cell surface. Pentamer analysis showed that only HBc18-27TCR transduced T cells expressed the appropriate α and β chainsrecognized by the HBc18-27-A201 specific pentamer and that the α and βchains were properly paired on the cell surface. No staining wasdetected in Mock transduced cells because PBL came from a healthy donorwhich was HLA-A2 negative.

FIG. 7 (C) shows the mean frequency of Vβ8+ T cells from 5 healthy, 5HBeAg+ chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg-chronicHBV patients (HBV DNA<10⁶ copies/ml). This indicates that the transducedVβ8 TCR chain is expressed equally well in chronic HBV patients andhealthy donors.

FIG. 7 (D) shows the mean frequency of HBc18-27-A201 specific pentamer+T cells from 5 healthy, 5 HBeAg+ chronic HBV patients (HBV DNA>10⁷copies/ml), and 5 HBeAg− chronic HBV patients (HBV DNA<10⁶ copies/ml).This indicates that the properly paired transduced TCR is expressedequally well in chronic HBV patients and healthy donors.

FIG. 8 (A) shows the FACS analysis of T cells transduced with HBc18-27A201 TCR to confirm that the introduced HBc18-27 TCR was functional.HBc18-27 TCR transduced or mock transduced T cells were co-culturedovernight with HLA-A2+ T2 cells which were first pulsed with HBc18-27peptide (“peptide”) and incubated for 5 h. The function of the T cellswas then assessed by intracellular cytokine staining for IFN-γ (leftcolumn), TNF-α (middle column) and IL-2 (right column). In the absenceof peptide, HBc18-27 TCR transduced cells were not activated (top row).Mock transduced cells were not activated by peptide pulsed T2 cells(middle row). HBc18-27 TCR transduced cells were stimulated to produceIFN-γ, TNF-α, and IL-2 following co-culture with peptide pulsed T2 cells(bottom row). Both CD8+ and CD8− (CD4+ T cells) were capable ofproducing all three cytokines confirming function of the introduced TCRin CD4+ T cells and multifunctional capacity of TCR re-directed cells.

FIG. 8 (B) shows that HBc18-27 TCR transduced T cells from chronic HBVpatients are equally functional to healthy donors. Mean frequency ofIFN-γ, TNF-α and IL-2 positive cells from 5 healthy, 5 HBeAg+ chronicHBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg− chronic HBV patients(HBV DNA<10⁶ copies/ml) are shown. The results show that T cellstransduced with HBc18-27-A201 TCR become multifunctional.

FIG. 9 shows the affinity of HBc18-27 TCR transduced T cells fromhealthy and chronic HBV patients compared to original C183 T cell clone.

FIG. 9 (A) shows the results when IFN-γ+ CD8+ HBc18-27 TCR transduced Tcells and C183 T cell clones were co-cultured with T2 cells pulsed withincreasing concentrations of HBc18-27 peptide for 5 h and then stainedfor IFN-γ. Data is presented as mean percentage of maximum response forHBc18-27 TCR transduced T cells derived from 5 healthy donors, 5 HBeAg+chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg− chronic HBVpatients (HBV DNA<10⁶ copies/ml) where C183 is 100% specific while TCRtransduced cell frequency varies. Results show that the affinity for theviral epitope of CD8+ HBc18-27 TCR transduced T cells derived frompatient T cells was identical when compared to the original C183 T cellclone and recognized the epitope down to concentrations of 0.1-1 pg/ml.

FIG. 9 (B) shows the results when IFN-γ+ CD4+ HBc18-27 TCR transduced Tcells and C183 T cell clones were co-cultured with T2 cells pulsed withincreasing concentrations of HBc18-27 peptide for 5 h and then stainedfor IFN-γ. Data is presented as mean percentage of maximum response forHBc18-27 TCR transduced T cells derived from 5 healthy donors, 5 HBeAg+chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg− chronic HBVpatients (HBV DNA<10⁶ copies/ml) where C183 is 100% specific while TCRtransduced cell frequency varies. CD4+ HBc18-27 TCR transduced cellsderived from patient T cells were less sensitive compared to C183 but50% of HBc18-27 specific T cells were activated at 100 pg/ml indicatingthat the introduced HBc18-27 TCR was highly sensitive, even in theabsence of the CD8 co-receptor.

FIG. 10 shows the expression of optimized HBc18-Opt-TCR compared to thewild type HBc18-WT-TCR.

FIG. 10 (A) shows histograms with the percentage of Vβ8+ T cells frommock, HBc18-WT-TCR and HBc18-Opt-TCR transduced PBL cells. Frequency ofVβ8+ cells was comparable between the optimized and wild type HBc18-TCRtransduced cells.

FIG. 10 (B) shows the comparison of the expression level of Vβ8 in mock,HBc18-WT-TCR and HBc18-Opt-TCR transduced T cells. To compare the levelof expression, mean fluorescent intensity (MFI) for Vβ8+ cells was used.The results show that there was no significant difference in the levelof Vβ8 expression between optimized and wild type TCR transduced cells.

FIG. 10 (C) shows the results obtained from HBc18-27 HLA-A2 specificpentamer staining. Cells from each group mentioned above were stainedwith anti-CD8 and HBc18-27 HLA-A2 specific pentamers. Nearly a two-foldincrease in the frequency of pentamer positive cells in T cellstransduced with HBc18-Opt-TCR constructs were shown compared to the wildtype TCR suggesting that the Vα3 chain may be expressed more efficientlyfrom the codon optimized construct compared to the wild type sequence.

FIG. 11 shows that there is an increased frequency of IFN-γ positivecells in T cells transduced with HBc18-Opt-TCR compared to HBc18-WT-TCRconstructs. Mock transduced T cells, HBc18-WT-TCR and HBc18-Opt-TCR werestimulated overnight with HLA-A2 positive T2 cells which were initiallypulsed or not pulsed with HBc18-27 peptide, in the presence of 2 μg/mlbrefeldin A. T cells were then labelled with anti-CD8 PE-Cy7 and stainedfor IFN-γ by intracellular cytokine staining. The results demonstratedthat there was nearly a two fold increase in the frequency of IFN-γpositive T cells transduced with the HBc18-Opt-TCR compared to T cellstransduced with the HBc18-WT-TCR. The increase in pentamer positivecells correlated with an increase in functional cells able to respond topeptide loaded target T cells.

FIG. 12 (A) EBV transformed B cells expressing HLA-A0201, -A0206 or-A0207 subtypes were loaded with increasing concentrations of HBVgenotype A/D HBc18-27 epitope (sequence provided in Table 1) andco-cultured with HBc-18-27 TCR transduced T cells for 5 h. T cellactivation was measured by intracellular cytokine staining for IFN-γ.The results show that the HBc-18-27 TCR transduced T cells recognize theHBc18-27 eptiope presented by multiple HLA-A2 subtypes.

FIG. 12 (B) EBV transformed B cells expressing HLA-A0201, -A0206 or-A0207 subtypes were loaded with increasing concentrations of HBVgenotype B/C HBc18-27 epitope (sequences provided in Table 1) andco-cultured with HBc-18-27 TCR transduced T cells for 5 h. T cellactivation was measured by intracellular cytokine staining for IFN-γ.The results show that the HBc-18-27 TCR transduced T cells recognize theHBc18-27 eptiope presented by multiple HLA-A2 subtypes.

FIG. 12 (C) show the results when HLA-A2+ T2 cells were loaded with eachof the mutant peptides of HBc18-27 epitope and co-cultured withHBc-18-27 TCR transduced T cells for 5 h. T cell recognition of themutant peptides was determined by intracellular cytokine staining forIFN-γ. The sequences of the mutant peptides are provided in Table 1. Ascan be seen from the results, the HBc-18-27 TCR transduced T cellsrecognized various mutants of the HBc18-27 eptiope. The Unstim 1 andUnstim 2 refer to the control where the HLA-A2+ T2 cells were not loadedwith any mutant peptide.

FIG. 13 (A) TCR transduced T cells from each patient group (healthy,HBeAg(−), HBeAg(+)) were co-cultured with DiOC labeled HepG2 cellsexpressing the entire HBV genome (HBV-HepG2) or the parental controlcell line (Ctrl-HepG2) at Effector:Target ratio of 1:1 for 6 h in thepresence of Propidium Iodide (PI). Dying target cells were DiOC+/PI+.Results are representative of 3 experiments. Effector T cells wereconsidered to be IFN-γ+/CD8+ cells determined by intracellular cytokinestaining after stimulation with peptide pulsed T2 cells. The resultsshowed that HBc18-27 TCR transduced T cells can kill HCC cell lineswhich endogenously express HBV proteins or are loaded with the HBV 18-27peptide.

FIG. 13 (B) DiOC labeled HCC cell lines (HepG2, SNU-387, SNU-368) wereloaded with increasing concentrations of HBc18-27 peptides andco-cultured with HBc-18-27 TCR transduced T cells at Effector to targetratio of 1:1 for 6 h in the presence of Propidium Iodide (PI). Dyingtarget cells were DiOC+/PI+. Results are representative of 3experiments. Effector T cells were considered to be IFN-γ+/CD8+ cellsdetermined by intracellular cytokine staining after stimulation withpeptide pulsed T2 cells. The results showed that HBc18-27 TCR transducedT cells are able to kill multiple HLA-A2+ HCC cell lines.

FIG. 14 (A) shows that the E10-1D T cell clone or HBs370-79 TCRtransduced T cells, recognize HBs370-79 epitope loaded HLA-A2+ T2 cells.E10-1D T cell clone is made according to the method provided in Example10. T2 cells were loaded with increasing concentrations of HBs370-79peptide and co-cultured with E10-1D or HBs370-79 TCR transduced T cellsfor 5 h and stained for IFN-γ. Data presented as percent maximumresponse to normalize differing frequency of each line. The results showthat HBs370-79 specific TCR is functional.

FIG. 14 (B) shows that HBs370-79 TCR transduced T cells recognizegenotypic variants of the HBs370-79 epitope (The sequences of thegenotypic variants are provided in Table 2). HBs370-79 TCR transduced Tcells were co-cultured with HLA-A2+ T2 cells loaded with 1 μg/ml peptideof each genotype for 5 h and activation was assessed by CD107a labelingand staining for IFN-γ. Genotype A & D sequences are identical. Theresults show that HBs370-79 specific TCR recognizes genotypic variantsof the HBs370-79 epitope.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are forconvenience listed in the form of a list of references and added at theend of the examples. The whole content of such bibliographic referencesis herein incorporated by reference.

DEFINITIONS

For convenience, certain terms employed in the specification, examplesand appended claims are collected here.

The term “comprising” is herein defined to be that where the variouscomponents, ingredients, or steps, can be conjointly employed inpracticing the present invention. Accordingly, the term “comprising”encompasses the more restrictive terms “consisting essentially of” and“consisting of:”

With the term “consisting essentially of” it is understood that theexogenous TCR polypeptide and/or polynucleotide according to theinvention “substantially” comprises the indicated sequence as“essential” element. Additional sequences may be included at the 5′ endand/or at the 3′ end. Accordingly, a polypeptide “consisting essentiallyof” sequence X will be novel in view of a known polypeptide accidentallycomprising the sequence X.

With the term “consisting of” it is understood that the polypeptideand/or polynucleotide according to the invention corresponds to at leastone of the indicated sequence (for example a specific sequence indicatedwith a SEQ ID Number or a homologous sequence or fragment thereof).

The term “exogenous T cell receptor” (TCR) is herein defined as arecombinant TCR which is expressed in a cell by introduction ofexogenous coding sequences for a TCR. In particular, the HBVepitope-reactive TCR may be expressed in a cell in which the TCR iseither not natively expressed or is expressed at levels that areinsufficient to induce a response by the cell or a responder cell uponTCR ligand binding.

The term “fragment” is herein defined as an incomplete or isolatedportion of the full sequence of the HBV epitope-reactive exogenous TCRwhich comprises the active site(s) that confers the sequence with thecharacteristics and function of the HBV epitope-reactive exogenous TCR.In particular, it may be shorter by at least one nucleotide or aminoacid. More in particular, the fragment comprises the active site(s) thatenable the HBV epitope-reactive exogenous TCR to recognise the HBc18-27epitope and/or the HBs370-79 epitope.

The term “HBV epitope-reactive T Cell Receptor (TCR)” is herein definedas a TCR which binds to an HBV epitope in the context of a MajorHistocompatibility Complex (MHC) molecule to induce a helper orcytotoxic response in the cell expressing the recombinant TCR. Inparticular, the HBV epitope may be HBc18-27. More in particular, the HBVepitope may comprise the sequence of SEQ ID NO:25. The HBV epitope maybe HBs370-79. More in particular, the HBV epitope may comprise thesequence of SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58.

The term “HBc 18-27 epitope” is herein defined as an epitope that canstimulate HLA class I restricted T cells. It may be used interchangeablyin the present invention as HBc18, HBc18-27, HBc18-27 peptide and thepeptide. The sequence of the epitope may be “FLPSDFFPSV” (SEQ ID NO:25).In the present invention, the term HBc18-27 is used to refer to theHBc18-27 epitope of genotype A/D prevalent amongst Caucasians ofsequence SEQ ID NO:25 unless otherwise stated. The region of the T cellreceptor that binds to the epitope is referred to as HBc18-27 TCR orHBc18 TCR.

The term “HBs370-79 epitope” is herein defined as an epitope that canstimulate HLA class I restricted T cells. The sequence of the epitopemay be “SIVSPFIPLL” (SEQ ID NO:56). In the present invention, the termHBs370-79 is used to refer to the HBs370-79 epitope of genotype A/Dprevalent amongst Caucasians of sequence SEQ ID NO:56 unless otherwisestated. The region of the T cell receptor that binds to the epitope isreferred to as HBs370-79 TCR.

The term “immunotherapeutically effective amount” is herein defined asan amount which results in an immune-mediated prophylactic ortherapeutic effect in the subject, i.e., that amount which will preventor reduce symptoms at least 50% compared to pre-treatment symptoms orcompared to a suitable control.

The term “isolated” is herein defined as a biological component (such asa nucleic acid, peptide or protein) that has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, i.e., other chromosomal and extrachromosomal DNA and RNA, andproteins. Nucleic acids, peptides and proteins which have been isolatedthus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids.

The term “operably connected” herein defined as a functional linkagebetween regulatory sequences (such as a promoter and/or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the regulatory sequences direct transcription of the nucleicacid corresponding to the second sequence.

The term “mutant” or “mutant form” of a TCR epitope is herein defined asone which has at least one amino acid sequence that varies from at leastone reference virus-encoded sequence via substitution, deletion oraddition of at least one amino acid, but retains the ability to bind andactivate the TCR bound and activated by the non-mutated epitope. Inparticular, the mutants may be naturally occurring or may berecombinantly or synthetically produced.

The term “soluble TCR” is herein defined as a soluble (secreted) form ofthe membrane bound TCR which is the molecule that is responsible for theT cell's recognition of the antigen for which it is specific. In itssoluble form it is analogous to a monoclonal antibody except that itrecognizes fragments of peptides associated with MHC molecules whileantibodies recognize determinants on a whole protein. In particular, theisolated polypeptide of the present invention may be used for producinga soluble TCR using any method known in the art. More in particular, thesoluble TCR may bind to the HBc18-27 and/or HBs370-79 epitope of HBV.

The term “subject” is herein defined as vertebrate, particularly mammal,more particularly human. For purposes of research, the subject mayparticularly be at least one animal model, e.g., a mouse, rat and thelike. In particular, for the animal models, the sequence of the TCR α-and β-chains may be selected based on species. In some cases, transgenicanimals expressing human MHC molecules may also be useful in evaluatingspecific aspects of the present invention.

A person skilled in the art will appreciate that the present inventionmay be practised without undue experimentation according to the methodgiven herein. The methods, techniques and chemicals are as described inthe references given or from protocols in standard biotechnology andmolecular biology text books.

In one aspect of the present invention, there is provided at least oneisolated cell comprising at least one HBV epitope-reactive exogenous Tcell receptor (TCR) and/or fragment thereof. Such cells may be suitablefor use in adoptive transfer protocols to provide a particularlyeffective mode of treatment. The isolated cells of the present inventionmay circumvent the problem of deletion and sub-optimal function ofHBV-specific CD8+ and CD4+ cells which may be present in patients withchronic HBV infection. In particular, the TCR from a patient whoresolved the HBV infection may be used to redirect the specificity ofthe lymphocytes of chronic HBV patients for TCR transfer.

In particular, HBV-reactive TCRs may be prepared by transforming ortransducing at least one isolated cell with one or more polynucleotidesencoding functional α- and/or β-chains and/or polypeptides of functionalα- and/or β-chains that may assemble to form at least one functionalHBV-epitope reactive TCR. In particular, the isolated cell may beisolated using any technique known in the art. More in particular, atleast one cell isolation kit, Ficoll-Paque density gradientcentrifugation, BD FACSAria Cell Sorting System and the like may be usedto isolate the cell.

The isolated cell may be at least one autologous cell, i.e., they may bederived from at least one subject that may receive the resultanttransduced or transformed cells. In particular, the isolated cells maybe derived from the peripheral blood lymphocytes and/or hematopoieticstem cells of the subject.

More in particular, the isolated cell may be at least one T cell thatinnately expresses at least one CD4+ cell surface marker, at least oneCD8+ cell surface marker, both CD4+ and CD8+ marker or neither CD4+ norCD8+ cell surface markers. In particular, the cell according to thepresent invention may be at least one T cell that also nativelyexpresses at least one TCR. The HBV-reactive exogenous TCR may bind thesame epitope as the natively expressed TCR, and/or may bind a differentepitope. In particular, the HBV-reactive exogenous TCR may be expressedin at least one cell in which the TCR may be either not nativelyexpressed or may be expressed at levels that may be insufficient toinduce response by the cell or responder cell upon TCR ligand binding.In particular, the isolated cell according to the present invention maybe transduced with two or more different HBV-reactive exogenous TCRs,i.e., TCRs which bind to two or more different HBV epitopes.

HBV-exposed patients who have cleared their viral infections, mayprovide excellent source of HBV-reactive T cells expressing highaffinity TCRs. In particular, HBV-epitope reactive TCRs may be preparedby isolating at least one HBV-reactive T cell from at least oneHBV-exposed aviremic individual, and cloning the polynucleotide sequenceencoding the α- and β-chains of the TCR from the HBV reactive T cell.Once these sequences have been cloned using standard methods known inthe art, the sequences may be delivered to at least one isolated celland the isolated cell may be incubated under conditions suitable forexpression of the TCR by the isolated cell. More in particular, thesuitable conditions may include standard cell culture conditions. Whenthe TCR is expressed in vitro or ex vivo, the cell expressing the TCRmay be evaluated for reactivity with HBV epitopes, among otherparameters of interest, as known in the art and as exemplified below. Insome protocols, i.e., those wherein the vector may be administered to atleast one subject, the TCR may also be expressed in vivo to provide atherapeutic effect in at least one subject in need thereof, i.e., atleast one subject with acute or chronic HBV infection or HBV-associatedcondition.

The HBV-reactive TCRs according to the present invention may befunctional in the isolated cell in which they may be expressed. Inparticular, they may be functional heterodimers of α and β TCR chainsassociated with a CD3 complex that recognizes at least one HBV epitopein the context of at least one Class I or Class II MHC molecule. Inhumans, the MHC restriction of at least one epitope may be dependent onat least one particular Human Leukocyte Antigen (HLA) expressed by atleast one cell presenting the antigen. HBV-reactive TCRs that mayrecognize HBV epitopes restricted to any HLA type (i.e., HLA-A, HLA-B,HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1)may be used in the present invention. For purposes of study, theHBV-reactive TCR may recognize at least one HBV epitope in the contextof at least one MHC molecule of at least one species other than human,e.g., H-2K of mouse.

In particular, the HBV-reactive TCR recognizes HBV epitopes that may beHLA-A2 restricted. Approximately 50% of the general population expressesthe MHC class I molecule HLA-A2, an HLA-A serotype. Therefore,HLA-A2-restricted TCRs may find widespread therapeutic use. Inparticular, the subtype may identify gene products of many HLA-A*02alleles, comprising HLA-A*0201, *0202, *0203, *0206, and *0207 geneproducts. There may be distinct differences in the subtypes betweenCaucasian and Asian populations. Whereas more than 95% of the HLA-A2positive Caucasian population is HLA-A0201 the HLA-A2 positive Chinesepopulation may be broken down into 23% HLA-A0201; 45% HLA-A0207; 8%HLA-A0206; 23% HLA-A0203.

The TCRs of the present invention may be HBV-epitope reactive. A list ofknown immunoreactive HBV epitopes and their sequences may be found onpage 233, chapter 11 (The effect of pathogens on the immune system:Viral hepatitis) of the book In immunodominance: The choice of theImmune System, J. A. Frelinger, ed (Weinheim: Wiley-VCH) which is hereinincorporated by reference. The HBV epitope may comprise at least onecore antigen, envelope antigen, surface antigen and/or mutants thereof.

In particular, the HBV epitope may comprise at least one hepatitis Bcore antigen, and/or mutants thereof. More in particular, the HBVepitope may comprise HBc18-27, and/or mutants thereof. The HBc18-27epitope may comprise “FLPSDFFPSV” (SEQ ID NO:25) or mutants thereof.Furthermore, the HBc18-27 epitope present in genotype A and D which maycomprise “FLPSDFFPSV” (SEQ ID NO: 25), which are most prevalent inEurope, differs from HBc18-27 epitope of genotypes B and C which maycomprise “FLPSDFFPSI” (SEQ ID NO: 26), which are most prevalent in Asia.

In particular, the HBV epitope may comprise at least one hepatitis Benvelope antigen, and/or mutants thereof. More in particular, the HBVepitope may comprise HBs370-79, and/or mutants thereof. The HBs370-79epitope may comprise “SIVSPFIPLL” (SEQ ID NO:56) or mutants thereof.Furthermore, the HBs370-79 epitope present in genotype A and D which maycomprise “SIVSPFIPLL” (SEQ ID NO:56), which are most prevalent inEurope, differs from HBs370-79 epitope of genotype B which may comprise“NILSPFMPLL” (SEQ ID NO: 57), and epitope of genotype C which maycomprise “NILNPFLPLL” (SEQ ID NO: 58), which are most prevalent in Asia.

In particular, the cell according to the present invention may furthercomprise at least one second HBV epitope-reactive exogenous TCR and/orfragment thereof, wherein the second HBV-reactive exogenous TCR may bethe same epitope as the first epitope or different from the first HBVepitope. In particular, the second HBV epitope may be HBs370-79 and thefirst epitope may be HBc18-27.

The exogenous TCR may comprise at least one α-chain comprising at leastone amino acid sequence selected from the group consisting the sequencesSEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 and/or mutants thereof. Inparticular, the exogenous TCR comprises at least one α-chain comprisingthe sequences SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. More inparticular, the α-chain comprises all three sequences, SEQ ID NO:9, SEQID NO:10 and SEQ ID NO:11. The α-chain may comprise the sequences of SEQID NO:9, SEQ ID NO:10 and SEQ ID NO:11 consecutively. In particular, theα-chain may consist essentially of the sequences SEQ ID NO:9, SEQ IDNO:10 and SEQ ID NO:11. All sequences used in the present invention aregiven in Tables 1 and 2.

The exogenous TCR may further comprise at least one β-chain comprisingat least one amino acid sequence selected from the group consisting ofthe sequences SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 and/or mutantsthereof. In particular, the β-chain may comprise all three sequences,SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In particular, the β-chainmay comprise the sequences of SEQ ID NO:21, SEQ ID NO:22 and SEQ IDNO:23 consecutively. More in particular, the β-chain may consistessentially of the sequences SEQ ID NO:21, SEQ ID NO:22 and SEQ IDNO:23.

In particular, the exogenous TCR may comprise at least one α-chainhaving at least 80% amino acid identity to SEQ ID NO:12 or a fragmentthereof and/or at least one β-chain having at least 80% amino acididentity to SEQ ID NO:24 or a fragment thereof. In particular, theα-chain and β-chain may be at least about 85%, at least about 90%, or atleast about 95% identical to SEQ ID NO:12 and SEQ ID NO:24 respectively.More in particular, the α-chain may comprise all sequences SEQ ID NO:9,SEQ ID NO:10 and SEQ ID NO:11 and β-chain may comprise all sequences SEQID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

The exogenous TCR may comprise at least one α-chain comprising at leastone amino acid sequence selected from the group consisting the sequencesSEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46 and/or mutants thereof. Inparticular, the exogenous TCR comprises at least one α-chain comprisingthe sequences SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46. More inparticular, the α-chain comprises all three sequences, SEQ ID NO:44, SEQID NO:45 and SEQ ID NO:46. The α-chain may comprise the sequences of SEQID NO:44, SEQ ID NO:45 and SEQ ID NO:46 consecutively. In particular,the α-chain may consist essentially of the sequences SEQ ID NO:44, SEQID NO:45 and SEQ ID NO:46. All sequences used in the present inventionare given in Tables 1 and 2.

The exogenous TCR may further comprise at least one β-chain comprisingat least one amino acid sequence selected from the group consisting ofthe sequences SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54 and/or mutantsthereof. In particular, the β-chain may comprise all three sequences,SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54. In particular, the β-chainmay comprise the sequences of SEQ ID NO:52, SEQ ID NO:53 and SEQ IDNO:54 consecutively. More in particular, the β-chain may consistessentially of the sequences SEQ ID NO:52, SEQ ID NO:53 and SEQ IDNO:54.

In particular, the exogenous TCR may comprise at least one α-chainhaving at least 80% amino acid identity to SEQ ID NO:47 or a fragmentthereof and/or at least one β-chain having at least 80% amino acididentity to SEQ ID NO:55 or a fragment thereof. In particular, theα-chain and β-chain may be at least about 85%, at least about 90%, or atleast about 95% identical to SEQ ID NO:47 and SEQ ID NO:55 respectively.More in particular, the α-chain may comprise all sequences SEQ ID NO:44,SEQ ID NO:45 and SEQ ID NO:46 and β-chain may comprise all sequences SEQID NO:52, SEQ ID NO:53 and SEQ ID NO:54.

The sequences encoding the α-chain and β-chain have been determined tobe the amino acid sequence for at least one productively rearrangedα-chain and β-chain respectively of a TCR reactive against HBV epitopeHBc18-27 and/or HBs370-79.

More in particular, the exogenous TCR reactive against HBV epitopeHBc18-27 may comprise at least one α-chain of SEQ ID NO:12 and/or atleast one (3-chain of SEQ ID NO:24. Even more in particular, theexogenous TCR reactive against HBV epitope HBc18-27 may consistsessentially of at least one α-chain of SEQ ID NO:12 and at least oneβ-chain of SEQ ID NO:24. The exogenous TCR reactive against HBV epitopeHBc18-27 may consists of at least one α-chain of SEQ ID NO:12 and atleast one β-chain of SEQ ID NO:24. In particular, the exogenous TCRreactive against HBV epitope HBc18-27 may consist of two α-chains andtwo β-chains.

More in particular, the exogenous TCR reactive against HBV epitopeHBs370-79 may comprise at least one α-chain of SEQ ID NO:47 and/or atleast one β-chain of SEQ ID NO:55. Even more in particular, theexogenous TCR reactive against HBV epitope HBs370-79 may consistsessentially of at least one α-chain of SEQ ID NO:47 and at least oneβ-chain of SEQ ID NO:55. The exogenous TCR reactive against HBV epitopeHBs370-79 may consists of at least one α-chain of SEQ ID NO:47 and atleast one β-chain of SEQ ID NO:55. In particular, the exogenous TCRreactive against HBV epitope HBs370-79 may consist of two α-chains andtwo β-chains.

Percentage identity may be determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. 87: 2264-68 (1990), modified Proc.Natl. Acad. Sci. 90: 5873-77 (1993)). Such algorithm is incorporatedinto the BLASTx program, which may be used to obtain amino acidsequences homologous to a reference polypeptide. The present inventionmay also encompass TCR α- or β-chains having amino acid sequencesincluding conservative amino acid substitutions. Such substitutions arewell known in the art.

According to another aspect, the present invention provides at least oneisolated polynucleotide comprising at least one sequence encoding atleast one α-chain and/or at least one sequence encoding at least oneβ-chain wherein, the encoded α-chain and β-chain may be part of at leastone HBV epitope-reactive TCR.

In particular, the sequence encoding the α-chain comprises at least onesequence selected from SEQ ID NO:1 and SEQ ID NO:5, at least onesequence selected from SEQ ID NO:2 and SEQ ID NO:6 and at least onesequence selected from SEQ ID NO:3 and SEQ ID NO:7 and/or the sequenceencoding the β-chain comprises at least one sequence selected from SEQID NO:13 and SEQ ID NO:17, at least one sequence selected from SEQ IDNO:14 and SEQ ID NO:18 and at least one sequence selected from SEQ IDNO:15 and SEQ ID NO:19. More in particular, the sequence encoding theα-chain may comprise SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 and/or thesequence encoding the β-chain may comprise SEQ ID NO:17, SEQ ID NO:18and SEQ ID NO:19.

In particular, the sequence encoding the α-chain of the HBc-18-27epitope-reactive TCR may have at least 80% sequence identity to SEQ IDNO:4 or SEQ ID NO:8 and/or the sequence encoding the β-chain of theHBc-18-27 epitope-reactive exogenous TCR may have at least 80% sequenceidentity to SEQ ID NO:16 or SEQ ID NO:20. In particular, the sequence(s)encoding the α-chain and β-chain may be at least about 85%, at leastabout 90%, or at least about 95% identical to SEQ ID NO:4 or SEQ ID NO:8and SEQ ID NO:16 or SEQ ID NO:20 respectively. More in particular, thesequence encoding the α-chain may be selected from the group consistingof SEQ ID NO:4 and SEQ ID NO:8, and/or the sequence encoding the β-chainmay be selected from the group consisting of SEQ ID NO:16 and SEQ IDNO:20. The HBc-18-27 epitope-reactive exogenous TCR may comprise twoα-chains encoded by the sequence SEQ ID NO:8 and two β-chains encoded bySEQ ID NO:20. More in particular, the α-chain and β-chain may be encodedby nucleotide sequences that comprise the consecutive sequence shown inSEQ ID NO:4 or SEQ ID NO:8 and SEQ ID NO:16 or SEQ ID NO:20respectively.

Even more in particular, the exogenous TCR may consists essentially ofat least one α-chain encoded by SEQ ID NO:4 or SEQ ID NO:8 and at leastone β-chain encoded by SEQ ID NO:16 or SEQ ID NO:20. The exogenous TCRmay consist of at least one α-chain encoded by SEQ ID NO:4 or SEQ IDNO:8 and at least one β-chain encoded by SEQ ID NO:16 or SEQ ID NO:20.In particular, the exogenous TCR may consist of two α-chains encoded bySEQ ID NO:4 or SEQ ID NO:8 and two β-chains encoded by SEQ ID NO:16 orSEQ ID NO:20.

In particular, the sequence encoding the α-chain comprises SEQ ID NO:40,SEQ ID NO:41 and SEQ ID NO:42 and/or the sequence encoding the β-chaincomprises SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.

More in particular, the sequence encoding the α-chain of the HBs370-79epitope-reactive exogenous TCR may have at least 80% sequence identityto SEQ ID NO:43 and the β-chain of the HBs370-79 epitope-reactiveexogenous T cell receptor has at least 80% sequence identity to SEQ IDNO:51. In particular, the sequence(s) encoding the α-chain and β-chainmay be at least about 85%, at least about 90%, or at least about 95%identical to SEQ ID NO:43 and/or SEQ ID NO:51 respectively. More inparticular, the sequence encoding the α-chain may be SEQ ID NO:43 andSEQ ID NO:51, and/or the sequence encoding the β-chain may be SEQ IDNO:51. The HBs370-79 epitope-reactive exogenous TCR may comprise twoα-chains encoded by the sequence SEQ ID NO:43 and two β-chains encodedby SEQ ID NO:51. More in particular, the α-chain and β-chain may beencoded by nucleotide sequences that comprise the consecutive sequenceshown in SEQ ID NO:43 and SEQ ID NO:51 respectively.

According to another aspect, the present invention provides at least oneisolated polypeptide encoded by the polynucleotides of the presentinvention.

According to still another aspect, the present invention provides atleast one isolated polypeptide comprising at least one amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10and SEQ ID NO:11. In particular, the polypeptide may have at least 80%amino acid identity to SEQ ID NO:12. More in particular, the polypeptidemay be at least about 85%, at least about 90%, or at least about 95%identical to SEQ ID NO:12.

The present invention also provides at least one isolated polypeptidecomprising at least one sequence selected from the group consisting ofSEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46. In particular, thepolypeptide may have at least 80% amino acid identity to SEQ ID NO:47.More in particular, the polypeptide may be at least about 85%, at leastabout 90%, or at least about 95% identical to SEQ ID NO:47.

The polypeptide may further comprise at least one sequence selected fromthe group consisting of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. Inparticular, the further sequence may have at least 80% amino acididentity to SEQ ID NO:24. More in particular, the further sequence maybe at least about 85%, at least about 90%, or at least about 95%identical to SEQ ID NO:24. Even more in particular, the polypeptide maycomprise the consecutive sequence of amino acids shown in SEQ ID NO:12and SEQ ID NO:24 respectively.

The polypeptide may further comprise at least one sequence selected fromthe group consisting of SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54. Inparticular, the further sequence may have at least 80% amino acididentity to SEQ ID NO:55. More in particular, the further sequence maybe at least about 85%, at least about 90%, or at least about 95%identical to SEQ ID NO:55. Even more in particular, the polypeptide maycomprise the consecutive sequence of amino acids shown in SEQ ID NO:47and SEQ ID NO:55 respectively.

The polypeptide according to the present invention may be at least oneHBV epitope-reactive exogenous TCR. In particular, the polypeptide maybe functionally equivalent to a specifically exemplified TCR sequencethat may have been modified by single or multiple amino acidsubstitutions, by addition and/or deletion of amino acids, or where oneor more amino acids have been chemically modified, but whichnevertheless retains the binding specificity and high affinity bindingactivity of the TCR protein of the present invention to the HBV epitope.In particular, the HBV epitope may be HBc18-27, HBs370-79 or a mutantthereof. More in particular, the polypeptide may be at least one solubleTCR. Even more in particular, the soluble TCR protein may lack theportions of the native cell-bound TCR and may be stable in solution(i.e., it does not generally aggregate in solution when handled asdescribed herein and under standard conditions for protein solutions).

The soluble TCR may be prepared by any method known in the art. Examplesof processes that may be used to prepare the soluble TCR may comprisebut are not limited to constructing polymeric receptor chains in whichan immunoglobulin heavy chain variable region from at least onephosphorylcholine-specific antibody may be substituted with TCR α and βvariable regions, introducing translational termination codons upstreamof the TCR transmembrane region or replacing the transmembrane domainsof the TCR α and β chain cDNAs with a signal for glycosylphosphatidylinositol (GPI) linkage from the carboxy terminus of the GPI linkedprotein Thy-1.

The soluble TCR may be linked to at least one anti-viral drug. Theanti-viral drug may target HBV. For example, anti-viral drugs maycomprise, but are not limited to, adefovir dipivoxil, interferonalfa-2b, pegylated interferon alfa-2a, lamivudine, entecavir,telbivudine and the like.

In particular, the soluble TCR may be linked to the anti-viral drugs toform soluble TCR-drug conjugates by any means known in the art. The linkmay be a chemical moiety comprising a covalent bond or a chain of atomsthat covalently attaches a soluble TCR to a drug moiety. Linkers maycomprise a divalent radical such as an alkylene, an arylene, aheteroarylene, moieties such as: —(CR2)nO(CR2)n-; repeating units ofalkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, caproamide andthe like.

According to one aspect, the present invention provides at least oneconstruct comprising the polynucleotide of the present inventionoperably connected to at least one promoter. The coding sequences for α-and β-chains of the TCR may be operably connected to at least onepromoter functional in the isolated cell. Suitable promoters may beconstitutive and inducible promoters, and the selection of anappropriate promoter may be well within the skill in the art. Forexample, suitable promoters may comprise, but are not limited to, theretroviral LTR, the SV40 promoter, the CMV promoter and cellularpromoters (e.g., the β-actin promoter).

According to another aspect, the present invention provides at least onevector comprising the construct according to the present invention orthe polynucleotide according to the present invention. In particular,the vectors may comprise, but not limited to, lentiviral vectors,retroviral vectors, adenoviral vectors, adeno-associated virus vectorsand Herpes Simplex Virus vectors. More in particular, retroviral vectorsmay be used for delivery of the constructs either in vitro, ex vivo orin vivo, as described in the examples.

According to still another aspect, the present invention provides atleast one T cell comprising the vector, the construct or the polypeptideaccording to the present invention.

According to one aspect, the present invention provides at least onemethod of preparing at least one T cell comprising at least one HBVepitope-reactive exogenous TCR for delivery to at least one subjectcomprising transducing at least one T cell isolated from the subjectwith the construct of and/or the vector of the present invention.Constructs and vectors according to the present invention may bedelivered to cells in vitro, ex vivo or in vivo using any number ofmethods known to those of skill in the art. For example, if the cellsare in vitro or ex vivo, they may be transformed or transduced accordingto standard protocols, e.g., those described in Molecular Cloning: ALaboratory Manual, 3d ed., Sambrook and Russell, CSHL Press (2001),incorporated herein by reference. Examples of methods may comprise butare not limited to, the CaCl₂ chemical method, electroporation and thelike. In particular, the constructs according to the present inventionmay be delivered into the cells in viva Suitable methods of delivery ofpolynucleotide constructs are known in the art, and may comprise but arenot limited to, viral vectors, nanoparticles, gold particles, lipoplexesand/or polyplexes.

According to another aspect, the present invention provides at least onemethod of preparing at least one HBV epitope-reactive exogenous TCRcomprising:

-   -   (a) isolating at least one HBV-epitope reactive T cell from at        least one HBV-exposed individual that resolved HBV infection;    -   (b) cloning at least one polynucleotide sequence encoding at        least one α- and/or β-chain of at least one TCR from the        HBV-epitope reactive T cell of step (a);    -   (c) delivering the polynucleotide sequence of step (b) to at        least one cell; and    -   (d) incubating the cell under conditions suitable for expression        of the HBV epitope-reactive exogenous T cell receptor by the        cell.

In particular, the HBV-epitope reactive T cell of step (a) mentionedabove may be HBc18-27 epitope reactive and/or HBs370-79 reactive.

According to one aspect, the present invention provides at least onecell, at least one vector and/or at least one polypeptide according toall aspects of the present invention, for use in the treatment of HBVinfection and/or HBV-related hepatocellular carcinoma.

According to another aspect, the present invention provides at least onemethod of treating HBV and/or inhibiting reactivation of HBV in at leastone subject comprising administering to the subject at least oneimmunotherapeutically effective amount of cells, at least one vectorand/or at least one polypeptide according to any aspect of the presentinvention.

A further possible application of the HBV-TCR redirected approach is inpatients with hepatocellular carcinoma (HCC). Chronic infection with HBVis the most important risk factor for the development of this livertumour and integration of HBV-DNA into hepatocytes frequently occurs intumour transformed cells. The therapy of HCC by conventionalchemotherapy, radiation or surgical resection presents severelimitations. HBV-specific T cells have the potential to recognize andkill HCC cells expressing HBV antigens and adoptive transfer ofHBV-TCR-redirected T cells may have the potential to obtain regressionof HCC. Such results were obtained in nasopharyngeal carcinoma, a tumourexpressing an Epstein-Barr Virus (EBV) protein, where infusion ofEBV-specific CD8+ cells showed promising results. Vectors comprisingpolynucleotides encoding TCRs, and/or cells comprising at least oneHBV-epitope reactive TCR may thus be useful in the treatment of HCC.

Accordingly, the present invention provides at least one method oftreating HBV-related hepatocellular carcinoma in at least one subjectcomprising administering to the subject at least oneimmunotherapeutically effective amount of cells, at least one vectorand/or at least one polypeptide according to any aspect of the presentinvention.

Vectors comprising polynucleotides encoding TCRs, and/or cellscomprising at least one HBV-epitope reactive TCR prepared as describedabove, may be suitably administered to a subject to treat acute orchronic HBV infection or conditions (including, e.g., hepatocellularcarcinoma) in the subject.

The cells expressing HBV-epitope reactive TCRs, vectors comprisingpolynucleotides encoding HBV-epitope reactive TCR and/or the polypeptideencoding the HBV-epitope reactive TCR may be prophylacticallyadministered to at least one subject to inhibit reactivation of HBVinfection. In particular, vectors of the invention may be administeredto cells from at least one subject ex vivo. More in particular, modes ofadministration of polynucleotide and/or viral vectors will be those thatspecifically and/or predominantly deliver the TCR coding sequences to Tcells and/or hematopoietic stem cells. In the case of a retroviralvector, it may be anticipated that suitable dosages will range fromabout 0.1 μg/10⁶ cells to about 10 μg/10⁶ cells, such as in the rangefrom about 1 μg/10⁶ cells to about 5 μg/10⁶ cells. More in particular,such dosages may prevent or reduce HBV-related symptoms at least 50%compared to pre-treatment symptoms or compared to a suitable control.Treatment with at least one retroviral vector according to the presentinvention may palliate or alleviate HBV infection and/or at least oneassociated condition, and/or may reduce incidence of progression tochronic HBV-associated conditions, without providing a cure. Inparticular, treatment may be used to cure or prevent an acute or chronicHBV infection or an associated condition, including hepatocellularcarcinoma.

In particular, the quantity of cells that make up theimmunotherapeutically effective amount of cells to be administereddepends on the subject to be treated. This may be dependent on but notlimited to, the capacity of the individual's immune system to mountTCR-mediated immune response, the age, sex and weight of the patient andthe severity of the condition being treated. The number of variables inregard to at least one individual's prophylactic or treatment regimenmay be large, and a considerable range of doses may be expected. Inparticular, cells may be administered in at least one amount from 5×10⁵cells/kg body weight to 1×10¹⁰ cells/kg body weight. More in particular,5×10⁶ cells/kg body weight to 1×10⁸ cells/kg body weight may beadministered. The maximal dosage of cells and/or viral vector to beadministered to the subject may be the highest dosage that does notcause undesirable and/or intolerable side effects. Suitable regimens forinitial administration and additional treatments may also becontemplated and may be determined according to conventional protocols.

According to another aspect, the present invention provides at least oneuse of at least one cell, at least one vector according and/or at leastone polypeptide according to any aspect of the present invention in thepreparation of a medicament for treating HBV infection and/orHBV-related hepatocellular carcinoma.

Suitable solid or liquid medicament preparation forms may be, forexample, granules, powders, tablets, coated tablets, (micro) capsules,suppositories, syrups, emulsions, suspensions, creams, aerosols, dropsor injectable solutions in ampule form and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavourings, sweeteners orsolubilizers are customarily used as described above. The medicamentsmay be suitable for use in a variety of drug delivery systems.

According to one aspect, the present invention provides at least one invitro method for diagnosing at least one subject that is able to resolveHBV infection, the method comprising:

-   -   (a) providing at least one sample from at least one subject;    -   (b) detecting the presence of at least one polynucleotide        comprising, substantially consisting of or consisting of at        least one nucleic acid sequence selected from SEQ ID NO: 4, SEQ        ID NO: 8, SEQ ID NO: 16 and SEQ ID NO: 20, SEQ ID NO: 43, SEQ ID        NO: 51, a homologue and/or a fragment thereof; and/or    -   (c) detecting the presence of at least one polypeptide        comprising, substantially consisting of or consisting of the        amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO:        47, SEQ ID NO: 55, a homologue and/or a fragment thereof;        wherein the presence of the polynucleotide and/or the        polypeptide is indicative of the subject being able to resolve        the HBV infection.

According to yet another aspect, the present invention provides at leastone in vitro method for diagnosing at least one subject as having or asbeing at risk of having HBV infections and/or HBV related hepatocellularcarcinoma, the method comprising:

-   -   (a) providing at least one sample from at least one subject;    -   (b) detecting the presence of at least one polynucleotide        comprising, substantially consisting of or consisting of at        least one nucleic acid sequence selected from SEQ ID NO: 4, SEQ        ID NO: 8, SEQ ID NO: 16 and SEQ ID NO: 20, SEQ ID NO: 43, SEQ ID        NO: 51, a homologue and/or a fragment thereof; and/or    -   (c) detecting the presence of at least one polypeptide        comprising, substantially consisting of or consisting of the        amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO:        47, SEQ ID NO: 55, a homologue and/or a fragment thereof;        wherein the absence of the polynucleotide and/or the polypeptide        correlates with the likelihood of the subject as having or as        being at risk for having HBV-infection and/or HBV-related        hepatocellular carcinoma.

In particular, the test sample may be urine, blood, serum, sweat, and/ororal fluid samples. More in particular, the presence of at least onepolynucleotide and/or polypeptide of the present invention may bedetermined using several methods known in the art. Some of the methodsmay comprise, but are not limited to, real time PCR, flow cytometry, PCRand/or geometric mean fluorescence intensity (MFI).

According to still another aspect, the present invention provides atleast one kit for detecting and/or treatment of HBV infection and/orHBV-related hepatocellular carcinoma, the kit comprising at least onecell, at least one vector and/or at least one polypeptide according toany aspect of the present invention. In particular, the kit may compriseat least one manual providing instruction on how to use the kit.

TABLE 1Sequences of HBV eptiope reactive exogenous TCR alpha 3 and beta 8 chains and naturally occurring HBc 18-27 epitope mutant peptides. SEQ ID NameSequence NO: CDR1 alpha 3 (WT) AAAACTAGTATAAACAATTTA 1 CDR2 alpha 3 (WT)TTAATACGTTCAAATGAAAGAGAG 2 CDR3 alpha 3 (WT)TGTGCTACGTGGCTCTCTGGTTCTGCAAGGCAACTGACCTTT 3 TCR alpha 3 (WT)ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTT 4CAACTGGCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGTTCCTTGTTGATCACGGCTTCCCGGGCAGCAGACACTGCTTCTTACTTCTGTGCTACGTGGCTCTCTGGTTCTGCAAGGCAACTGACCTTTGGATCTGGGACACAATTGACTGTTTTACCTGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATG ACGCTGCGGCTGTGGTCCAGCTGACDR1 alpha 3 (OPT) AAGACATCAATCAACAACTTG 5 CDR2 alpha 3 (OPT)CTGATTCGGAGTAATGAGCGGGAA 6 CDR3 alpha 3 (OPT)TGTGCTACATGGCTGAGTGGCAGCGCACGGCAATTGACTTTT 7 TCR alpha 3 (OPT)ATGGAGACCCTTCTGGGAGTGTCCCTCGTGATTCTGTGGCTG 8CAGCTTGCTCGGGTGAATTCTCAGCAGGGCGAGGAAGACCCGCAGGCCCTTAGCATTCAGGAAGGGGAGAACGCTACCATGAATTGCTCATACAAGACATCAATCAACAACTTGCAGTGGTACCGTCAGAACTCTGGGAGAGGACTCGTGCACCTGATCCTGATTCGGAGTAATGAGCGGGAAAAACACTCTGGAAGGCTGAGGGTGACCCTCGATACCTCTAAAAAATCCTCCTCCCTGCTGATAACCGCCAGCAGGGCCGCCGACACCGCTTCCTACTTCTGTGCTACATGGCTGAGTGGCAGCGCACGGCAATTGACTTTTGGGAGTGGCACTCAGCTGACAGTGCTGCCCGACATCCAGAATCCAGATCCCGCAGTGTATCAGCTGAGAGACTCAAAGTCAAGTGACAAGAGTGTGTGCCTGTTCACTGATTTTGACTCTCAGACCAACGTCTCTCAGTCTAAGGACAGCGACGTTTACATCACTGACAAAACTGTGCTGGACATGCGCAGTATGGACTTTAAATCAAATTCCGCCGTGGCTTGGAGCAATAAGTCTGACTTCGCCTGTGCTAATGCTTTTAATAACTCCATCATTCCGGAGGATACATTTTTCCCTAGCCCCGAGTCATCCTGCGACGTGAAGCTGGTGGAGAAGTCATTCGAGACCGACACCAATCTTAACTTTCAGAACCTGTCCGTTATCGGGTTTAGAATCCTGCTGCTGAAGGTTGCCGGATTCAACCTGCTTATG ACGTTGCGCCTGTGGTCCAGCTGACDR1 alpha 3 KTSINNL 9 CDR2 alpha 3 LIRSNERE 10 CDR3 alpha 3CATWLSGSARQLTF 11 TCR alpha 3 METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMN12 CSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATWLSGSARQLTFGSGTQLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVDMRSMDFKSNSAVAWSNKSDFACANALFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF RILLLKVAGFNLLMTLRLWSS.CDR1 beta 8 (WT) ATT TCA GGA CAC GAC TAC CTT 13 CDR2 beta 8 (WT)TAC TTT AAC AAC AAC GTT CCG ATA 14 CDR3 beta 8 (WT)TGT GCC AGC AGC AAT CGG GCG AGC TCC TAC 15 AAT GAG CAG TTC TTCTCR beta 8 (WT) ATGGACTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTG 16GTAGCAAAGCACACAGATGCTGGAGTTATCCAATCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGCAATCGGGCGAGCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGA AAGGATTCCAGAGGCTAGCDR1 beta 8 (OPT) ATC TCT GGG CAC GAC TAC CTG 17 CDR2 beta 8 (OPT)TAT TTT AAT AAC AAT GTG CCT ATC 18 CDR3 beta 8 (OPT)TGT GCC TCC TCC AAC CGG GCC TCC TCT TAT 19 AAC GAG CAG TTC TTCTCR beta 8 (OPT) ATGGACAGCTGGACACTGTGCTGCGTGAGCCTGTGCATTCTG 20GTGGCCAAGCACACCGACGCCGGCGTGATCCAGAGCCCTCGCCACGAGGTGACCGAAATGGGCCAGGAGGTGACACTGCGCTGCAAGCCAATCTCTGGGCACGACTACCTGTTCTGGTACAGGCAGACCATGATGAGGGGCCTGGAACTGCTGATCTATTTTAATAACAATGTGCCTATCGATGACTCTGGCATGCCCGAGGACAGGTTCTCCGCCAAGATGCCCAACCCAGCTTCTCCACCCTGAAGGATCCAGCCCTCCGAACCTAGGGACTCCGCCGTGTACTTCTGTGCCTCCTCCAACCGGGCCTCCTCTTATAACGAGCAGTTCTTCGGCCCTGGAACCCGCCTGACCGTGCTGGAGGACCTGAAAAATGTGTTTCCCCCCGAGGTGGCCGTGTTTGAACCAAGCGAGGCCGAGATCAGCCACACACAGAAGGCCACCCTGGTGTGTCTGGCCACCGGATTCTATCCCGATCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAGGAGGTGCACTCTGGCGTGAGCACCGACCCTCAGCCACTGAAAGAGCAGCCCGCCCTGAATGATTCTCGGTACTGCCTGTCCAGCCGCCTGCGCGTGTCTGCCACCTTCTGGCAGAACCCCAGAAATCACTTCAGGTGCCAGGTGCAGTTCTATGGGCTGAGCGAGAACGACGAATGGACCCAGGACAGAGCCAAGCCTGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGACTGCGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCCGCCACAATTCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGCGG AAAGACTCCCGGGGCTGACDR1 beta 8 ISGHDYL 21 CDR2 beta 8 YFNNNVPI 22 CDR3 beta 8CASSNRASSYNEQFF 23 TCR beta 8 MDSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRC24 KPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSNRASSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDSRGHBc18-27 peptide FLPSDFFPSV 25 (HBV genotype A/D/E/F) (WT C18-27)HBc18-27 peptide FLPSDFFPS I 26 (HBV genotype B/C)(27I)Natural Variant 1 FLPNDFFPSV 27 Natural Variant 2 FLP N DFFPS A 28Natural Variant 3 FLP A DFFPS I 29 Natural Variant 4 FLP V DFFPS I 30Natural Variant 5 FLP T D Y FPSV 31 Natural Variant 6 FLPSDF Y P P V 32Natural Variant 7 (26G) FLPSDFFP G V 33 Natural Variant 8 (25M, FLPSDFFMG V 34 26G) Natural Variant 9 (23Y) FLPSD Y FPSV 35Natural Variant 10 (24L) FLPSDF L PSV 36 Natural Variant 11 (21P) FLP PDFFPSV 37 Natural Variant 12 (27A) FLPSDFFPS A 38Natural Variant 13 (24Y) FLPSDF Y PSV 39

TABLE 2Sequences of HBV eptiope reactive exogenous TCR alpha 12 and beta 7.8chains and naturally occurring HBe 370-79 epitope mutant peptides.SEQ ID Name Sequence NO: CDR1 alpha 12 (WT) GAC CGA GGT TCC CAG TCC 40CDR2 alpha 12 (WT) ATA TAC TCC AAT GGT 41 CDR3 alpha 12 (WT)TGT GCC GTG AAC CTC TAT GCA GGC AAC ATG 42 CTC ACC TTT TCR alpha 12 (WT)ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTT 43CAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACCTCATGCAGGCAACATGCTCACCTTTGGAGTGGGGAACAAGGTTAATGGTCAAACCCCATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTC ATGACGCTGCGGCTGTGGTCCAGCTGACDR1 alpha 12 DRGSQS 44 CDR2 alpha 12 IYSNG 45 CDR3 alpha 12CAVNLYAGNMLTF 46 TCR alpha 12 MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIAS47 LNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLYAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLLMTLRLWSSCDR1 beta 7.8 (WT) TCG GGT CAT GTA TCC 48 CDR2 beta 7.8 (WT)TTC CAG AAT GAA GCT CAA 49 CDR3 beta 7.8 (WT)TGT GCC AGC AGC TCG GAC TTT GGC AAT CAG  50 CCC CAG CAT TTTTCR beta 7.8 (WT) ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTA 51GGGACAGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGCACACAGCAGGAGGACTCCGCCGTGTATCTCTGTGCCAGCAGCTCGGACTTTGGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAG GATTTCTGA CDR1 beta 7.8 SGHVS52 CDR2 beta 7.8 FQNEAQ 53 CDR3 beta 7.8 CASSSDFGNQPQHF 54 TCR beta 7.8MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRC 55DPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSSDFGNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAV LVSALVLMAMVKRKDFHBs370-79 peptide SIVSPFIPLL 56 (HBV genotype A/D) HBs370-79 peptideNILSPFMPLL 57 (HBV genotype B) HBs370-79 peptide NILNPFLPLL 58(HBV genotype C)

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

EXAMPLES

Standard molecular biology techniques known in the art and notspecifically described were generally followed as described in Sambrookand Russel, Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (2001).

The following examples are provided to assist in a further understandingof the invention. The particular materials and conditions employed areintended to be further illustrative of the invention and are notlimiting upon the reasonable scope of the appended claims.

Example 1 Cloning Hepatitis B Virus Specific T Cell Specific for HBVCore 18-27 Epitope (HBc18-27)

Peripheral blood lymphocytes (PBL) were isolated from fresh bloodobtained from a HBV patient who was able to resolve the disease and whowas of HLA haplotype HLA-A0201 herein referred to as “a resolvedHLA-A201 HBV patient”.

The blood was first heparinised and PBL were isolated using Ficoll-Paquedensity gradient centrifugation. PBL were then washed three times inHanks Balanced Salt Solution (HBSS) (Invitrogen, Carlsbad, Calif.),resuspended in Aim-V medium, 2% human AB serum (Invitrogen, Carlsbad,Calif.), stimulated with 1 μM of HBV core 18-27 epitope (“HBc18-27peptide”; FLPSDFFPSV; SEQ ID NO: 25; Primm SRL, Milano, Italy) plus 20U/ml interleukin-2-(R&D systems, Minneapolis, Minn.) and plated in a24-well plate at 4×10⁶ cells/well for 10 days.

The HBc18-27-specific CD8+ T cells prepared as above were then labelledwith phycoerythrin (PE)-conjugated HLA-A2 class I pentamers hereinreferred to as “HBc18-27-HLA-A201 pentamers” and/or “HLA-A2 pentamer”(Proimmune, Oxford, United Kingdom) bearing the HBc18-27 epitope for 30min at 37° C. and purified via magnetic cell sorting using anti-PEmicrobeads (Miltenyi Biotech, Surrey, United Kingdom). Followingisolation, cells were labelled with Cy-chrome-conjugated anti-CD8+ (BDPharmingen, San Diego, Calif.) and the percentage of stained T cellswere determined by Fluorescence-activated cell sorting (FACS) analysisusing a FACScan flow cytometer (BD Biosciences, SanDiego, Calif.) andanalyzed using CellQuest software (BD Biosciences). The flow cytometrymethod used is further described in Gehring et al., incorporated hereinby reference. The results are shown in FIG. 2A.

The CD8+ and HBc18-HLA-A201 pentamer positive T cells as mentionedabove, were then cloned using the limiting dilution assay as describedin “Current Protocols in Immunology” Copyright © 2007 by John Wiley andSons, Inc., and expanded in Aim-V 2% AB serum (Invitrogen, Carlsbad,Calif.), 20 U/ml IL-2, 10 ng/ml IL-7, and 10 ng/ml IL-15 (R&D systems,Minneapolis, Minn.) with 1.5 μg/ml phytohemagglutinin (Sigma-Aldrich,Dorset, United Kingdom) using allogeneic irradiated PBL as feeder cells.Cells were plated at 1 cell/well on 96 well plates and wells positivefor T cell growth were tested for HBc18-27 reactivity by intracellularcytokine staining for IFN-γ according to the protocol provided inGehring et al., incorporated herein by reference.

Highly positive wells for peptide reactivity were then screened usingthe specific HBc18-27 HLA-A201 pentamer (Proimmune, Oxford, UnitedKingdom) and a T cell clone, referred to herein as C183 was selected forfurther study. The results, depicted in FIG. 2A, demonstrate that 100%of CD8+ and HBc18-27-HLA-A201 pentamer-reactive T cells were detected.Clonality was tested using a panel of T cell receptor (TCR) Vβmonoclonal antibodies (Beckman Coulter, Fullerton, Calif.). Thisprocedure involved the use of a panel of Vβ antibodies that stained allknown Vβ chain family members and where positive staining for only oneVβ chain meant that it was highly likely that all of the T cells of thatclone expressed the same TCR. Accordingly, the resultant TCR Vβdistribution for C183, shown in FIG. 2B demonstrates that all the Tcells from C183 were Vβ8 positive suggesting homogeneity (likely thatthey were all derived from a single cell) and thus clonality.

C183 thus was shown to be a clonal, CD8+, HBc18-27 specific cytotoxic Tcell clone and was grown and maintained in Aim-V, 2% AB serum, 20 U/mlIL-2, 10 ng/ml IL-7, and 10 ng/ml IL-15 (R&D Systems, Abingdon, UnitedKingdom) in a 5% CO₂ humidified incubator at 37° C.

Example 2 Recognition of Tumor Cells and Primary Human Hepatocytes byC183 T Cell Clone

HepG2 is a HLA-A2+, hepatocyte-like tumor cell line isolated from aprimary hepatocellular carcinoma tumor (HCC). HepG2 cells (American TypeCulture Collection, Rockford, Md.) were grown and maintained in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, 0.5 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/mlstreptomycin, MeM amino acids, L-glutamine, MeM nonessential amino acids(Invitrogen, Carlsbad, Calif.), and 5 μg/ml Plasmocin (InvivoGen, SanDiego, Calif.) to prevent mycoplasma contamination. A derivative of thisline, HepG2.105 (Kindly provided by Michael Nassal at the University ofFreiberg) was engineered to express the entire HBV genome under thecontrol of a doxycycline dependent regulator, which permits viralantigen to be expressed at various levels as described in Sun andNassal, 2006. A vector control parental cell line, HepG2TA2-7 (Kindlyprovided by Michael Nassal at the University of Freiberg) was stablytransfected with the doxycycline dependent regulator but not the HBVgenome. Both cell lines HepG2.105 and HepG2TA2-7, were grown inDulbecco's modified Eagle's medium supplemented with 10%tetracycline-approved FBS (BD Biosciences, San Diego, Calif.), 20 mMHEPES, 0.5 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/mlstreptomycin, MeM nonessential amino acids (Invitrogen), 200 μg/ml G418sulfate, 80 μg/ml Hygromycin B (AutogenBioclear, Wiltshire, UnitedKingdom). Doxycycline (0.1 to 100 ng/ml, BD Biosciences) was added tocultures to regulate HBV expression.

The standard HepG2 cells were labeled with 0.75 μM carboxyfluoresceinsuccinimidyl ester (CFSE; Invitrogen) for 10 min at 37° C. Cells werewashed, and CFSE-labelled targets were pulsed with 10⁻⁸ M HBc18-27peptide (Primm SRL, Milano, Italy) for 1 h on ice. Unlabelled (0 MHBc18-27 peptide) and CFSE-labelled (plus HBc18-27 peptide) HepG2 cellswere mixed together in a 1:1 ratio, 2×10⁵ cells of each HepG2 cell line,in 24-well plates and incubated overnight to permit HepG2 adherence. Todetermine if C183 was capable of killing HCC tumor cells, C183 clonesobtained from Example 1 were co-cultured with the HepG2 cells that wereadhered to the wells (“targets”). A total of 4×10⁵ of C183 clones wereadded to the targets and incubated for 5 h at 37° C. The resultanteffector (C183):target (unlabelled+CFSE-labelled HepG2 cells) ratio was1:1. After 5 h co-incubation, HepG2 cells were collected viatrypsinization and cytotoxicity was determined by comparing ratio ofpeptide pulsed targets to unpulsed targets in wells +/− the addition ofC183. The results shown in FIG. 3 depict that CFSE negative cells (noHBc18-27 peptide) which were co-cultured with C183 remained and nearlyall HepG2 cells which were HBc18-27 and CFSE positive were eliminated byC183 as shown in the last column. The results indicate that the HBc18-27specific cytotoxic T cell clone, C183 was capable of killing tumor cellspresenting HBV peptides.

Further, to determine if C183 can recognize endogenously processedantigen presented by HCC cells, C183 was co-cultured with HepG2.105 orHepG2TA2-7 control line and then tested for cytokine production andcytotoxic degranulation (CD107a+ staining), an indirect measure ofkilling. 1×10⁵ cells of each HepG2.105 and HepG2TA2-7 cell line wereseeded separately into 96-well plates and incubated overnight to permitHepG2 adherence. For cytotoxic degranulation assays, CD107a-PE antibody(BD Pharmingen, San Diego) was first added to all wells before a totalof 1×10⁵ of C183 clones were added to each well with the adheredHepG2.105 or HepG2TA2-7 control line and incubated for 5 h at 37° C. Theresultant effector (C183):target (HepG2.105 or HepG2TA2-7 control line)ratio was 1:1. Following the incubation, C183 T cells were harvested,washed and stained with Cy-chrome conjugated anti-CD8+ (BD Pharmingen,San Diego, Calif.) then permeabilized and fixed using Cytofix/Cytoperm(BD Pharmingen, San Diego) according to the manufacturer's instructions.Cells were washed and incubated with PE-conjugated anti-human IFN-γantibody (R&D systems) for 30 min on ice then washed and analyzed byflow cytometry using the FACScan flow cytometer (BD Biosciences,SanDiego, Calif.) and analyzed using CellQuest software (BDBiosciences). The flow cytometry method used is further described inGehring et al. incorporated herein by reference.

As shown in FIG. 4A, C183 did not respond to the control cell lineHepG2TA2-7 that was HBV negative. However, as shown in FIG. 4B,co-culture of C183 with HepG2.105 cells expressing HBV, stimulated IFN-γproduction and degranulation, indicating that C183 clone recognized theHBc18-27 epitope endogenously processed and presented by hepatocellularcarcinoma cells.

In order to determine if C183 T cell clone can respond to naturallyinfected hepatocytes, primary hepatocytes were isolated from HLA-A2+ orHLA-A2-explanted chronic HBV livers and co-cultured with C183 accordingto the procedure described below.

Liver tissue was obtained from both HLA-A2+ or HLA-A2-explanted chronicHBV patients under informed consent according to ethical and moralguidelines of the institution. Tissue sections were collected inWilliam's E medium (Sigma-Aldrich, Dorset, United Kingdom) andmaintained at 4° C. for a maximum of 2 h before cell isolation.Hepatocytes were isolated by enzyme perfusion described for use withhuman liver by Strain et al. (Strain, Ismail et al., 1991) with somemodifications. Briefly, a section of liver of about 100 to 200 g wascut, and exposed vessels on a single surface were cannulated with 3-mminternal diameter tubing. The tissue was perfused sequentially at 50ml/min with 500 ml PBS-HEPES wash solution to remove William's E medium,500 ml PBS-HEPES-0.5 mM EGTA, and again with 500 ml PBS-HEPES washsolution (The solutions were obtained from Invitrogen Ltd.). Finally,300 ml enzyme solution (0.05% collagenase (Roche, Indianapolis, Ind.),0.012% hyaluronidase (Sigma, Dorset, United Kingdom), 0.025% Dispase II(Roche), 0.005% DNase (Roche) containing 5 mM CaCl₂), maintained at 41°C., was perfused with recirculation, and enzymatic digestion continuedfor 10 to 20 min until the liver was judged to be substantiallysoftened. Tissue was mechanically dissociated in 200 ml Hanks' balancedsalt solution (Invitrogen Ltd.) containing 10% FBS, 5 mM CaCl₂. The cellsuspension was filtered through a 60-μm cell strainer and pelleted at37×g for 10 min at 4° C. Cells were washed three times in Hanks'balanced salt solution, after which viability and yield were assessed.Purified hepatocytes were used immediately after isolation. 100,000primary hepatocytes/well were added to 96 well plates and 75,000 C183clones were added to respective wells for 5 h with anti-CD107a-PEantibody (BD Pharmingen, San Diego) for degranulation plus 10 μg/mlbrefeldin A (Sigma-Aldrich). Following the incubation, T cells wereharvested, washed and stained with Cy-chrome conjugated anti-CD8+ (BDPharmingen, San Diego, Calif.) then permeabilized and fixed usingCytofix/Cytoperm (BD Pharmingen, San Diego) according to themanufacturer's instructions. Cells were washed and incubated withFITC-conjugated anti-human IFN-γ antibody (R&D systems, Abingdon, UK)for 30 min on ice then washed and analyzed by flow cytometry using theFACScan flow cytometer (BD Biosciences, SanDiego, Calif.) and analyzedusing CellQuest software (BD Biosciences). The flow cytometry methodused is further described in Gehring et al. incorporated herein byreference.

The results in FIG. 5 show that C183 discharged cytotoxic granules,measured by CD107a staining, in response to HLA-A2 positive HBV infectedprimary hepatocytes but not to HLA-A2 negative HBV infected hepatocytesor HLA-A2+ uninfected hepatocytes, indicating that the C183 canrecognize naturally infected primary human hepatocytes.

Example 3 Recognition of Additional HLA-A2 Subtypes and Variant Peptides

Chronic HBV infection is predominantly an Asian problem and it is thusimportant to know if the HBc18-27 specific TCR can recognize HLA-A2subtypes that dominate in the Asian population. HBV has a total of fivegenotypes (A, B, C, D and G). The HBc18-27 epitope expressed by HBVgenotypes B and C (FLPSDFFPSI; SEQ ID NO:26) are the most prevalent inAsia. Genotypes A and D, most prevalent in Europe and United states,(FLPSDFFPSV; SEQ ID NO:25) are characterized by a valine at position 27whereas genotypes B and C are characterized by an isoleucine at position27. This can have a profound impact on the ability of HBV patients topresent the HBc18-27 epitope or it can affect TCR recognition of theepitope.

The ability of C183 to recognize HBc18-27 epitope from genotypes B andC, and A and D presented by different subtypes of the HLA-A2 MHC class Ifamily was determined using Epstein-Barr virus (EBV) transformed B cellswith known subtypes of HLA-A2. (Kindly provided by Professor Chan Soh Haat WHO Immunology and Training Research Centre, Singapore). EBVtransformed B cells from any other source may be used in this procedure.The EBV transformed B cells (10⁵ cells/well) were loaded with increasingconcentrations (1 pM-1000 pM) of HBc18-27 peptide from either HBVgenotypes A/D or HBV genotypes B/C for 1 h at 25° C. (HBV GenotypeA/D=FLPSDFFPSV; Primm SRL; HBV genotype B/C=FLPSDFFPSI, Genscript,Piscataway, N.J.) EBV B cells were washed with HBSS to remove excesspeptide and co-cultured with 7.5×10⁴ C183 for 5 h in the presence of 10ug/ml brefeldin A. IFN-γ production was measured by intracellularcytokine staining to determine T cell activation as described in Gehringet al. incorporated herein by reference.

The results are shown in FIG. 6. The results show the ability of C183 torecognize the HBc18-27 epitope from four different HBV genotypes (A, B,C and D) and that the HBc18-27 epitopes were presented by the three mostdominant HLA-A2 subtypes (HLA-A201, HLA-A206, and HLA-A207). The C183 Tcell response is plotted at % IFN-γ+ cells. The black bars represent thefrequency of IFN-γ+C183 T cells able to respond to the HBc18-27 epitopefrom HBV genotypes A/D (C18-V) whereas the grey bars represent thefrequency of C183 T cells able to respond to HBc18-27 epitope from HBVgenotypes B/C(C18-I) The data shows that HBc18-27 epitopes presented by(A) HLA-A201; (B) HLA-A206; and (C) HLA-A207 MHC class I molecules wererecognized by C183. C183 recognized the HBc18-27 epitope with highsensitivity (≦1 pM) from all genotypes and the epitope was recognizedalmost equally when presented by the three most dominant HLA-A2 subtypes(A) A0201, (B) A0206 and (C) A0207.

To further show that the HBc-18-27 TCR transduced T cells recognize theHBc18-27 eptiope presented by multiple HLA-A2 subtypes and variousmutants of the HBc18-27 epitope, EBV transformed B cells expressingHLA-A0201, -A0206 or -A0207 subtypes were loaded with increasingconcentrations of HBV genotype A/D HBc18-27 epitope (FIG. 12 (A)) andHBV genotype B/C HBc18-27 epitope, (FIG. 12 (B)) (sequence provided inTable 1) and co-cultured with HBc-18-27 TCR transduced T cells for 5 h.T cell activation was measured by intracellular cytokine staining forIFN-γ. The results as shown in FIGS. 12 (A) and (B) show that theHBc-18-27 TCR transduced T cells recognize the HBc18-27 eptiopepresented by multiple HLA-A2 subtypes.

FIG. 12 (C) shows the results when HLA-A2+ T2 cells were loaded witheach of the mutant peptides of HBc18-27 epitope and co-cultured withHBc-18-27 TCR transduced T cells for 5 h. T cell recognition of themutant peptides was determined by intracellular cytokine staining forIFN-γ. The sequences of the mutant peptides are provided in Table 1. Ascan be seen from the results, the HBc-18-27 TCR transduced T cellsrecognized various mutants of the HBc18-27 epitope. The Unstim 1 andUnstim 2 refer to the control where the HLA-A2+ T2 cells were not loadedwith any peptide.

Example 4 Isolation of C183 T Cell Receptor Alpha and Beta Chain DNA

Total RNA was isolated from 5×10⁶ C183 clones using TRIzol (Invitrogen).TCR alpha and beta chain cloning was performed on a contract basis byPrimm SRL (Milano, Italy) and supplied in Topo blunt II cloning vectorfor downstream cloning.

The sequence analysis revealed the presence of one TCR beta chain,Vβ8.2, and one TCR alpha chain, Vα3 in C183. Further DNA sequenceanalysis using the Immunogenetics V-Quest algorithm(http://imgt.cines.fr/IMGT_vquest/share/textes/) identified 3 uniqueComplementarity Determining Region (CDR) in the Vα3 chain (CDR1α: SEQ IDNO:9, CDR2α: SEQ ID NO:10, CDR3α: SEQ ID NO:11) and the three unique CDRin the Vβ8.2 chain (CDR1β: SEQ ID NO:21, CDR2β: SEQ ID NO:22, CDR3β: SEQID NO:23).

Example 5 Retroviral Constructs Containing HBc18-27 Specific TCR(HBc18-27 TCR) and Lymphocyte Transduction

TCR Vα3 and TCR Vβ8.2 cDNA prepared according to Example 4 above, werecloned individually into retroviral vector MP71 as described in Engels,Cam et al. 2003. (Kindly provided by Professor Hans Stauss, Royal Freeand UCL Medical School, London, UK) using a 5′ Not-1 site and 3′ BsrG1sites (New England Biolabs, Ipswich, Mass.). TCR Vα3, Vβ8.2 and MP71vector were digested for 1 h at 37° C. with 10 U of each enzyme.Digested products were isolated on 1% agarose gel by electrophoresis andpurified using Qiagen gel extraction kit. TCR chains and MP71 vectorwere mixed in 1:1 ratio and ligated overnight at 4° C. with T4 DNAligase (Promega) to form retroviral constructs-MP71-TCR Vα3 and MP71-TCRVβ8.2. These constructs were then sequenced to confirm DNA insert wascorrect.

To determine the expression and function of the cloned TCR, theconstructs were used for preparation of retroviral supernatant which wasfurther used for T cell transduction as explained below. Expression ofinserted alpha and beta chains was driven by viral long terminal repeats(LTR) found in each construct.

Retroviral supernatants were prepared using a transient transfectionmethod which comprised using Phoenix amphotropic packaging cell line(Clontech Laboratories, US) that was first plated at 2×10⁶ cells/dishand allowed to adhere for 24 h in Iscove's Modified Dulbecco's Medium(IMDM), 10% FBS, 25 mM HEPES, Glutamax (Invitrogen) and 5 ug/mlPlasmocin (Invivogen). Cells were transiently co-transfected using theCaCl₂ method for 24 h with 9 μg each of MP71-TCR Vα3, MP71-TCR Vβ8.2together with 6 μg of amphotropic envelope (kindly provided by ProfessorHans Stauss, Royal Free and UCL Medical School, London, UK) IMDM wasthen replaced with Aim-V 2% human AB serum and phoenix cells wereincubated for an additional 24 h before retroviral supernatants werecollected for transduction.

Healthy donor and chronic HBV patient peripheral blood lymphocytes (PBL)were used to test expression and functionality of the cloned HBc18-27TCR. Peripheral blood mononuclear cells (PBMC) were isolated fromvolunteers under informed consent by ficoll density gradientcentrifugation. PBMC were stimulated with 600 U/ml interleukin-2 (IL-2;R&D Systems) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego,Calif.) for 48 h. Untreated 24 well tissue culture plates were coatedwith 30 ug/ml Retronectin (Takara Bio, Otsu Shiga, Japan) overnight at4° C. Wells were then washed with HBSS and blocked with PBS 2% BSA for30 min. Lymphocytes were harvested, washed, counted and 5×10⁵ cellsplated into retronectin coated wells and mixed with the retroviralsupernatants collected as described above. Mock transduced cells whichwere included as negative control, were cultured with the supernatantfrom Phoenix cells that were not transfected with retroviral vectors.IL-2 was added to wells to final concentration of 600 U/ml. Thelymphocytes were incubated for 24 h in the retroviral supernatant, afterwhich time the medium was replaced with Aim-V 2% human AB serum plus 100U/ml IL-2. Transduced and mock transduced cells were grown foradditional 3 days. After 3 days, the cell surface expression of the TCRwas measured in both the TCR transduced and mock transduced cells byimmunofluorescence staining. CD8-PE-Cy7 (BD Biosciences), Vβ8-PEexpression (Beckman Coulter) and HLA-A201-HBc18-PE pentamer (Proimmune)staining were quantified by flow cytometry. Flow cytometry was performedon stained cells using a FACs Canto flow cytometer (BD Biosciences), anddata were analyzed with the FACs Diva program (BD Biosciences).

As shown in FIG. 7A, expression of Vβ8 was significantly increased inHBc18-27 TCR transduced lymphocytes compared to mock transduced cells,which expressed only endogenous levels of Vβ8. Vβ8 expression wasobserved in both CD8+ and CD8− cells. To properly bind pentamer requiresthe cooperative interaction of a correctly paired, introduced alpha andbeta chain as well as the co-receptor CD8. The CD8+ HBc18-27 TCRtransduced T cells bound HBc18-27-HLA-A201 pentamers indicating that theVα3 TCR chain was also expressed and the introduced TCR was correctlypaired on the cell surface of transduced lymphocytes (FIG. 7B).

The HBc18-27 TCR transduction efficiency is confirmed in FIG. 7 (C)which show the mean frequency of Vβ8+ T cells from 5 healthy, 5 HBeAg+chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg− chronic HBVpatients (HBV DNA<10⁶ copies/ml). FIG. 7 (D) which shows the meanfrequency of HBc18-27-A201 specific pentamer+ T cells from 5 healthy, 5HBeAg+ chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5 HBeAg−chronic HBV patients (HBV DNA<10⁶ copies/ml) confirms that thetransduced HBc18-27 TCR was correctly paired and bound HBc18-27-HLA-A201pentamers. These data demonstrate that T cells from both healthy donorsand chronic HBV patients could be transduced with the HBc18 TCR withsimilar efficiency.

Example 6 Function of HBc18-27 TCR Transduced T Cells

To confirm that the introduced HBc18-27 TCR was functional, HLA-A2positive T2 cells (American Type Culture Collection (Rockford, Md.))were used to stimulate HBc18-27 TCR transduced or mock transducedlymphocytes. T2 cells were cultured in RPMI 1640 supplemented with 10%heat-inactivated fetal bovine serum (FBS), 20 mM HEPES, 0.5 mM sodiumpyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, MeM amino acids,Glutamax, MeM nonessential amino acids (Invitrogen Ltd) and 5 μg/mlPlasmocin (InvivoGen, San Diego, Calif.). The T2 cells were pulsed with1 μg/ml HBc18-27 peptide for 1 hr and then washed to remove excesspeptide.

In order to stimulate the HBc18-27 TCR transduced lymphocytes, referredto herein as HBc18-27 TCR transduced T cells, that were preparedaccording to Example 5, the HBc18-27 TCR transduced or mock transduced Tcells were co-cultured overnight with the two different types of T2cells (pulsed with HBc18-27 peptide and not pulsed) at 37° C. with 2ug/ml brefeldin A. The HBc18-27 TCR transduced and mock transduced Tcells were then washed and stained with anti-CD8-PE-Cy7 (BD Pharmingen,San Diego, Calif.) fixed and permeablized using cytofix/cytoperm (BDBiosciences) according to manufacturer's instructions. T cells were thenstained for TNF-α-Alexa 488, IL-2-PE and IFN-γ-APC (BD Biosciences) for30 min on ice. Cells were washed and analyzed by flow cytometry usingFACs Canto flow cytometer (BD Biosciences), and data were analyzed withthe FACs Diva program (BD Biosciences).

The results are shown in FIG. 8A. The mock transduced T cells failed toproduce any cytokines (i.e. IFN-γ, TNF-α or IL-2) in the presence orabsence of HBc18-27 epitope. HBc18-27 TCR transduced T cells did notrespond to T2 cells in the absence of peptide (FIG. 8A, row 1) butproduced IFN-γ, TNF-α and IL-2 in response to HBc18-27 peptide loaded T2cells (FIG. 8A, row 3) indicating that the introduced HBc18-27 TCR wasfunctional when expressed in primary T cells. Furthermore, theintroduced HBc18-27 TCR was functional in CD8− cells (CD4+ cells) aswell as CD8+, expanding its potential usefulness for immunotherapybecause CD4+ T cells provide additional IL-2 to maintain T cell survivaland co-stimulatory ligands such as CD40L, which has the potential toboost B cell responses.

FIG. 8 (B) shows that HBc18-27 TCR transduced T cells from chronic HBVpatients are equally functional to transduced T cells from healthydonors. Mean frequency of IFN-γ, TNF-α and IL-2 positive cells, as seenin FIG. 8A, from 5 healthy, 5 HBeAg+ chronic HBV patients (HBV DNA>10⁷copies/ml), and 5 HBeAg− chronic HBV patients (HBV DNA<10⁶ copies/ml)are shown. The results show that T cells transduced with HBc18-27-A201TCR become multifunctional with similar frequencies of cytokine positivecells in each patient group.

Example 7 Affinity of HBc18-27 TCR Transduced T Cells

Due to the possible decreased level of expression of the introducedHBc18-27 TCR, it was possible that the engineered T cells may have alower affinity for the actual viral epitope compared to the original Tcell clone, C183. To test this hypothesis, HLA-A2 positive T2 cells(American Type Culture Collection, Rockford, Md.) were pulsed withHBc18-27 peptide at concentrations ranging from 1 fg/ml to 1 μg/mlaccording to the protocol described in Example 6. Peptide loaded T2cells were co-cultured with TCR transduced T cells from 5 healthydonors, 5 HBeAg+ chronic HBV patients (HBV DNA>10⁷ copies/ml), and 5HBeAg− chronic HBV patients (HBV DNA<10⁶ copies/ml) and the original Tcell clone C183 for 5 h. T cell activation was measured by intracellularcytokine staining for IFN-γ. Results are displayed at percent of maximumIFN-γ response because the frequency of HBc18-27 TCR transduced cellsvaried among patients while C183 was always 100% specific for theHBc18-27 epitope. The results showed that CD8+ HBc18-27 TCR transduced Tcells displayed an affinity identical to C183 down to 1 pg/ml (FIG. 9A).

The affinity of HBc18-27 TCR transduced CD4+ T cells was determined inthe same assay. CD8− HBc18-27 specific cells were considered CD4+ Tcells because they were capable of responding to the HBc18-27 epitope inan antigen specific, dose dependent fashion. CD4+ HBc18-27 TCRtransduced T cells showed slightly lower affinity compared to C183. Thedifference in the affinity to the viral epitope between CD8+ and CD4+ Tcells may likely be due to CD8+ co-receptor binding, which stabilizesTCR interaction with the HLA-A2 molecule. The CD8+ co-receptor is absenton CD4+ T cells and therefore affinity is slightly reduced but stillhighly sensitive.

Example 8 Codon Optimization of HBc18-27 TCR DNA Sequence

Due to the presence of redundancy in the genetic code, most amino acidscan be coded for by multiple codons, which are used with differentfrequencies and efficiencies within organisms and can actually result inless efficient translation of proteins. Due to genetic variation and therecombination events that occur during T cell development, DNA encodingfor the HBc18-27 TCR may be comprised of codons that are not optimallytranslated within humans. Therefore, codon optimization for both theVβ8.2 and Vα3 chains of the HBc18-TCR was done to result in a moreefficient expression of HBc18-27 TCR and increased sensitivity andfunction (Service by Genscript, Piscataway, N.J.).

Codon optimized Vα3 and Vβ8.2 were cloned individually into MP71 vectorusing 5′Not-1 and 3′BsrG1 restriction enzyme sites as described above.In order to confirm that the codon optimized TCR, referred to herein asHBc18-Opt-TCR, was still capable of being expressed, PBL were transducedwith the HBc18-Opt-Vα3 and HBc18-Opt-Vβ8.2 TCR constructs as describedabove. For the HBc18-27 wild type TCR, referred to herein asHBc18-WT-TCR, PBL were also transduced with the HBc18-WT-Vβ8.2 (MP71-TCRVβ38.2 from example 7) and HBc18-WT-Vα3 (MP71-TCR Vα3 from example 7)for comparison. HBc18-27 transduced PBL were analyzed for Vβ8 expressionand the ability to bind HBc18-HLA-A201 pentamers (Proimmune, Oxford,United Kingdom) by flow cytometry 3 days after transduction. Resultsshowed that the frequency of Vβ8+ T cells was similar between PBLtransduced with HBc18-Opt-TCR (21.5%) compared to HBc18-WT-TCR (21.1%)transduced cells (FIG. 10A)

The amount of Vβ8 expressed in the HBc18-Opt-TCR transduced lymphocytesand HBc18-WT-TCR transduced lymphocytes was measured by meanfluorescence intensity (MFI) using the FACs Diva software (BDBiosciences) (FIG. 10B). Isotype values were subtracted to determinespecific MFI. The results showed that the amount of Vβ8 expressed wasalmost identical in both the HBc18-Opt-TCR transduced lymphocytes andHBc18-WT-TCR transduced lymphocytes (FIG. 10B).

To determine the ability to bind HBc18-HLA-A201 pentamers, theHBc18-Opt-TCR transduced lymphocytes and HBc18-WT-TCR transducedlymphocytes were labelled with anti-CD8-PE-Cy7 (BD Pharmingen, SanDiego, Calif.) and HBc18-HLA-A201 pentamers (Proimmune, Oxford, UnitedKingdom). The percentages of stained lymphocytes were determined byFluorescence-activated cell sorting (FACS) analysis using a FACs Cantoflow cytometer (BD Biosciences, SanDiego, Calif.) and analyzed usingFACs Diva software (BD Biosciences). The results showed that thefrequency of HBc18-HLA-A201 pentamer positive cells was two fold higherin cells transduced with HBc18-Opt-TCR than the HBc18-WT-TCR (FIG. 10C),suggesting that the Vα3 chain may be expressed more efficiently from thecodon optimized construct compared to the wild type sequence. BecauseVβ8 staining was equivalent in FIG. 10A increased pentamer binding islikely related to better expression of the Vα3 chain to increaseexpression of correctly paired HBc18-27 TCR on the cell surface.

Following confirmation that the HBc18-Opt-TCR was properly expressed inthe transduced T cells, we tested functionality of the HBc18-Opt-TCRcompared to the HBc18-WT-TCR. HBc18-Opt-TCR and HBc18-WT-TCR or mocktransduced T cells were stimulated overnight with HLA-A2 positive T2cells in the presence or absence of HBc18-27 peptide plus 2 μg/mlbrefeldin A according to the protocol described in Example 6. T cellswere then labelled with anti-CD8 PE-Cy7 (BD Pharmingen, San Diego,Calif.) and fixed and permeablized using cytofix/cytoperm according tomanufacturer's instructions (BD Biosciences). Cells were then stainedwith anti-IFN-γ-APC (BD Biosciences) for 30 min on ice and T cell IFN-γproduction was analyzed by flow cytometry.

The results show that there was nearly a two fold increase in thefrequency of IFN-γ positive T cells transduced with the HBc18-Opt-TCRcompared to T cells transduced with the HBc18-WT-TCR (FIG. 11). Mocktransduced cells were negative in the presence of absence of HBc18-27peptide. Therefore, the increase in pentamer positive cells correlatedwith an increase in functional cells able to respond to peptide loadedtargets.

Example 9 HBc18-27 TCR Transduced T Cells Kill HCC Cell Lines WhichEndogenously Express HBV Proteins or are Loaded with the HBV 18-27Peptide

HBc18-27 TCR transduced T cells from each patient group (healthy,HBeAg(−), HBeAg(+)) were co-cultured with DiOC labeled HepG2 cellsexpressing the entire HBV genome (HBV-HepG2) or the parental controlcell line (Ctrl-HepG2) at Effector:Target ratio of 1:1 for 6 h in thepresence of Propidium Iodide (PI). Dying target cells were DiOC+/PI+. Tcells were excluded from the analysis by staining with anti-CD11a-APC(BD Bioscience). Results are representative of 3 experiments. Effector Tcells were considered to be IFN-γ+/CD8+ cells determined byintracellular cytokine staining after stimulation with peptide pulsed T2cells. The results as shown in FIG. 13 (A) show that HBc18-27 TCRtransduced T cells from healthy donors or chronic HBV patients can killHCC cell lines which endogenously express HBV proteins. This suggeststhat HBc18 TCR transduced T cells could recognize tumor cells frompatients expressing the HBV core antigen.

To determine if TCR transduced T cells could kill multiple HLA-A2+ HCCcell lines, DiOC labeled HCC cell lines (HepG2 were obtained from ATCC;SNU-387, SNU-368 were obtained from the Korean cell line bank) wereloaded with increasing concentrations of HBc18-27 peptides andco-cultured with HBc-18-27 TCR transduced T cells at Effector to targetratio of 1:1 for 6 h in the presence of Propidium Iodide (PI). Dyingtarget cells were DiOC+/PI+. T cells were excluded from the analysis bystaining with anti-CD11a-APC (BD Bioscience). Results are representativeof 3 experiments. Effector T cells were considered to be IFN-γ+/CD8+cells determined by intracellular cytokine staining after stimulationwith peptide pulsed T2 cells. The results as shown in FIG. 13 (B) showedthat HBc18-27 TCR transduced T cells are able to kill multipleHLA-A2+HCC cell lines.

Example 10 Cloning Hepatitis B Virus Specific T Cell Specific for HBVEnvelope 370-79 Eptitope (HBs370-79)

Peripheral blood lymphocytes (PBL) were isolated and prepared accordingto the method of Example 1 and stimulated with 1 μM of HBV envelope370-79 epitope (“HBs370-79 peptide”; SIVSPFIPLL; SEQ ID NO: 56; PrimmSRL, Milano, Italy) plus 20 U/ml interleukin-2-(R&D systems,Minneapolis, Minn.) and plated in a 24-well plate at 4×10⁶ cells/wellfor 10 days.

The HBs370-79-specific CD8+ T cells were enriched using IFN-γ captureassay according to manufacturer's instructions. (Miltenyi Biotech,Surrey, United Kingdom).

The IFN-γ+HBs370-79− specific T cells were then cloned using thelimiting dilution assay as described in “Current Protocols inImmunology” Copyright © 2007 by John Wiley and Sons, Inc., and expandedin Aim-V 2% AB serum (Invitrogen, Carlsbad, Calif.), 20 U/ml IL-2, 10ng/ml IL-7, and 10 ng/ml IL-15 (R&D systems, Minneapolis, Minn.) with1.5 μg/ml phytohemagglutinin (Sigma-Aldrich, Dorset, United Kingdom)using allogeneic irradiated PBL as feeder cells. Cells were plated at 1cell/well on 96 well plates and wells positive for T cell growth weretested for HBs370-79 reactivity by intracellular cytokine staining forIFN-γ according to the protocol provided in Gehring et al. incorporatedherein by reference. T cell clone, referred to herein as E10-1D wasselected for further study.

E10-1D HBs370-79 specific cytotoxic T cell clone was grown andmaintained in Aim-V, 2% AB serum, 20 U/ml IL-2, 10 ng/ml IL-7, and 10ng/ml IL-15 (R&D Systems, Abingdon, United Kingdom) in a 5% CO₂humidified incubator at 37° C.

Example 11 Affinity and Function of HBs370-79 Specific TCR Transduced TCells and Recognition of Genotype Variant Peptides

Experiments similar to that described in Example 6 were carried out withHBs370-79 specific TCR transduced T cells. The E10-1D T cell clone orHBs370-79 TCR transduced T cells, recognize HBs370-79 epitiope loadedHLA-A2+ T2 cells. T2 cells were loaded with increasing concentrations ofHBs370-79 peptide and co-cultured with E10-1D or HBs370-79 TCRtransduced T cells for 5 h and stained for IFN-γ. Data presented aspercent maximum response to normalize differing frequency of each line.The results as shown in FIG. 14 (A) show that HBs370-79 specific TCR isfunctional but that HBs370 TCR transduced T cells were lower affinitythan the original T cell clone E10-1D.

Experiments similar to that described in Example 3 were also carried outwith HBs370-79 specific TCR transduced T cells. HBs370-79 TCR transducedT cells recognize genotypic variants of the HBs370-79 epitope (Thesequences of the genotypic variants are provided in Table 2). HBs370-79TCR transduced T cells were co-cultured with HLA-A2+ T2 cells loadedwith 1 μg/ml peptide of each genotype for 5 h and activation wasassessed by CD107a labeling and staining for IFN-γ. Genotype A & Dsequences are identical. The results as shown in FIG. 14 (B) show thatHBs370-79 specific TCR recognizes genotypic variants of the HBs370-79epitope.

REFERENCES

-   Engels, B., H. Cam, et al. (2003). “Retroviral vectors for    high-level transgene expression in T lymphocytes.” Hum Gene Ther    14(12): 1155-68.-   Gehring A. J et al. (2007). The Level of Viral Antigen Presented by    Hepatocytes Influences CD8 T-Cell Function. J Virol.; 81(6):    2940-2949.-   Karlin S. and Altschul S. F, (1990). Methods for assessing the    statistical significance of molecular sequence features by using    general scoring schemes. Proc Natl Acad Sci USA.; 87(6): 2264-2268.-   Maini, M. and Bertoletti A (2006) “The effect of pathogens on the    immune system: Viral hepatitis”. In immunodominance: The choice of    the Immune-   System, J. A. Frelinger, ed (Weinheim: Wiley-VCH). Molecular    Cloning: A Laboratory Manual, 3d ed., Sambrook and Russell, CSHL    Press (2001).-   Schumacher et. al., (2002), Nat Rev. Immunol, Vol 2: 512.-   Strain, A. J., et al. 1991. Native and recombinant human hepatocyte    growth factors are highly potent promoters of DNA synthesis in both    human and rat hepatocytes. J Clin Invest 87:1853-7.-   Sun, D. and M. Nassal (2006). “Stable HepG2- and Huh7-based human    hepatoma cell lines for efficient regulated expression of infectious    hepatitis B virus.” J Hepatol 45(5): 636-45.

1.-42. (canceled)
 43. An isolated cell comprising at least one of an HBVepitope-reactive exogenous T cell receptor and a fragment thereof,wherein the HBV epitope is HLA-A2 restricted.
 44. The cell according toclaim 43, wherein the HBV epitope-reactive exogenous T cell receptorreacts with an HBV epitope that comprises at least one of: (i) at leastone hepatitis B core antigen, (ii) at least one hepatitis envelopeantigen, and (iii) at least one mutant of (i) or (ii).
 45. The cellaccording to claim 43, wherein the HBV epitope comprises at least oneof: (i) an HBc18-27 epitope, (ii) an HBs370-79 epitope, and (iii) amutant of (i) or (ii).
 46. The cell according to claim 45, wherein theHBc18-27 epitope comprises at least one of SEQ ID NO:25 and SEQ IDNO:26, and wherein the HBs370-79 epitope comprises a sequence selectedfrom the group consisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO:58.
 47. The cell according to claim 43, wherein at least one of: (I) theHBV epitope reactive exogenous T cell receptor comprises at least oneα-chain comprising a sequence that is selected from: (a) SEQ ID NO:9,SEQ ID NO:10 and SEQ ID NO:11, or a mutant thereof, and (b) SEQ IDNO:44, SEQ ID NO:45 and SEQ ID NO:46, and a mutant thereof; and (II) theHBV epitope-reactive exogenous T cell receptor comprises at least oneβ-chain comprising a sequence that is selected from: (a) SEQ ID NO:21,SEQ ID NO:22 and SEQ ID NO:23 a or a mutant thereof, and (b) SEQ IDNO:52, SEQ ID NO:53 and SEQ ID NO:54 or a mutant thereof.
 48. The cellaccording to claim 43, wherein the exogenous T cell receptor compriseseither or both of: (a) at least one of (i) at least one α-chain havingat least 90% amino acid identity to SEQ ID NO: 12 or a fragment thereof,and (ii) at least one β-chain having at least 90% amino acid identity toSEQ ID NO:24 or a fragment thereof; and (b) at least one of (i) at leastone α-chain having at least 90% amino acid identity to SEQ ID NO: 47 ora fragment thereof, and (ii) at least one β-chain having at least 90%amino acid identity to SEQ ID NO:55 or a fragment thereof.
 49. Anisolated polynucleotide comprising at least one sequence encoding atleast one of a T cell receptor α-chain and a T cell receptor β-chain,wherein the α-chain and the β-chain are of at least one HBVepitope-reactive exogenous T cell receptor, and wherein the HBV epitopeis HLA-A2 restricted.
 50. The isolated polynucleotide according to claim49, wherein at least one of: (i) the sequence encoding the α-chaincomprises at least one sequence selected from SEQ ID NO:1 and SEQ IDNO:5, at least one sequence selected from SEQ ID NO:2 and SEQ ID NO:6,and at least one sequence selected from SEQ ID NO:3 and SEQ ID NO:7, and(ii) the sequence encoding the β-chain comprises at least one sequenceselected from SEQ ID NO:13 and SEQ ID NO:17, at least one sequenceselected from SEQ ID NO:14 and SEQ ID NO:18, and at least one sequenceselected from SEQ ID NO:15 and SEQ ID NO:19.
 51. The isolatedpolynucleotide according to claim 49, wherein at least one of: (i) thesequence encoding the α-chain comprises SEQ ID NO:5, SEQ ID NO:6 and SEQID NO:7, and (ii) the sequence encoding the β-chain comprises SEQ IDNO:17, SEQ ID NO:18 and SEQ ID NO:19.
 52. The isolated polynucleotideaccording to claim 49, wherein, the α-chain has at least 90% sequenceidentity to SEQ ID NO:4 or SEQ ID NO:8, and the β-chain has at least 90%sequence identity to SEQ ID NO:16 or SEQ ID NO:20.
 53. The isolatedpolynucleotide according to claim 49, wherein at least one of: (i) thesequence encoding the α-chain is selected from the group consisting ofSEQ ID NO:4 and SEQ ID NO:8, and (ii) the sequence encoding the β-chainis selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:20.54. The isolated polynucleotide according to claim 49, wherein at leastone of: (i) the sequence encoding the α-chain comprises a sequenceselected from SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, and (ii) thesequence encoding the β-chain comprises a sequence selected from SEQ IDNO:48, SEQ ID NO:49 and SEQ ID NO:50.
 55. The isolated polynucleotideaccording to claim 49, wherein, the α-chain of the HBV epitope-reactiveexogenous T cell receptor has at least 90% sequence identity to SEQ IDNO:43 and the β-chain of the HBV epitope-reactive exogenous T cellreceptor has at least 90% sequence identity to SEQ ID NO:51.
 56. Anisolated polypeptide comprising one or more of: (a) at least one of (i)an α-chain comprising SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and(ii) a β-chain comprising SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23;and (b) at least one of (i) an α-chain comprising SEQ ID NO:44, SEQ IDNO:45 and SEQ ID NO:46, and (ii) a β-chain comprising SEQ ID NO:52, SEQID NO:53 and SEQ ID NO:54; wherein the polypeptide is at least one HBVepitope-reactive exogenous T cell receptor and the HBV epitope is HLA-A2restricted.
 57. The isolated polypeptide according to claim 56 whereinat least one of: (a) the α-chain has at least 90% amino acid identity toSEQ ID NO:12 and the β-chain has at least 90% amino acid identity to SEQID NO:24; and (b) the α-chain has at least 90% amino acid identity toSEQ ID NO:47 and the β-chain has at least 90% amino acid identity to SEQID NO:55.
 58. The polypeptide according to claim 56, wherein the HBVepitope is at least one of (i) an HBc18-27 epitope, (ii) an HBs370-79epitope, and (iii) a mutant of (i) or (ii).
 59. The polypeptideaccording to claim 56, wherein the polypeptide is at least one soluble Tcell receptor (TCR) or a fragment thereof and is linked to at least oneanti-viral drug.
 60. A method for treating HBV or HBV-relatedhepatocellular carcinoma or for inhibiting reactivation of HBV in atleast one subject, comprising administering to the subject at least oneimmunotherapeutically effective amount of cells according to claim 43.61. An in vitro method for diagnosing at least one subject as being ableto resolve HBV infection or as having or being at risk for having HBVinfection or HBV related hepatocellular carcinoma, the methodcomprising: (a) providing at least one sample from at least one subject;and (b) detecting in the sample presence or absence of at least onepolynucleotide according to claim 49, wherein the presence of thepolynucleotide is indicative of the subject being able to resolve theHBV infection and the absence of the polynucleotide correlates withlikelihood that the subject has or is at risk for having HBV-infectionor HBV-related hepatocellular carcinoma.
 62. An in vitro method fordiagnosing at least one subject as being able to resolve HBV infectionor as having or being at risk for having HBV infection or HBV relatedhepatocellular carcinoma, the method comprising: (a) providing at leastone sample from at least one subject; and (b) detecting in the samplepresence or absence of at least one polypeptide according to claim 56,wherein the presence of the polypeptide is indicative of the subjectbeing able to resolve the HBV infection and the absence of thepolypeptide correlates with likelihood that the subject has or is atrisk for having HBV-infection or HBV-related hepatocellular carcinoma.